CA1267496A - Purification and activation of proteins from insoluble inclusion bodies - Google Patents
Purification and activation of proteins from insoluble inclusion bodiesInfo
- Publication number
- CA1267496A CA1267496A CA000517604A CA517604A CA1267496A CA 1267496 A CA1267496 A CA 1267496A CA 000517604 A CA000517604 A CA 000517604A CA 517604 A CA517604 A CA 517604A CA 1267496 A CA1267496 A CA 1267496A
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- CA
- Canada
- Prior art keywords
- protein
- sds
- solution
- ion
- inclusion bodies
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
Abstract
ABSTRACT OF THE DISCLOSURE
Purification and activation of proteins produced in transformant microorganisms as insoluble, biologically inactive inclusion bodies can be effected by solubilizing the inclusion bodies in SDS; dialyzing or refrigerating the protein solution to remove a portion of the SDS;
chromatographing the protein solution on an ion-retardation resin to remove the remaining SDS from the protein; and chromatographing the protein solution thus obtained on an anion-exchange resin.
Purification and activation of proteins produced in transformant microorganisms as insoluble, biologically inactive inclusion bodies can be effected by solubilizing the inclusion bodies in SDS; dialyzing or refrigerating the protein solution to remove a portion of the SDS;
chromatographing the protein solution on an ion-retardation resin to remove the remaining SDS from the protein; and chromatographing the protein solution thus obtained on an anion-exchange resin.
Description
~26~7gL~6 PP:570 FROM INSOLUBLE INCLU5ION BODIES
~AC K r R l~tJND OF THE INVEETION
This invention relates to methods for puriEying and activating proteins that are produced as insoluble, biologically inacti~e inclusion bodies in microorganisms that have been transformed with recombinant DNA
expression vectors to direct expression of the protein of interest.
Recombinant DNA technology allows the insertion of a vector carrying foreign (heterologous) DNA into a microorganism in a manner which allows the heterologous DNA to be expressed; that is, the vector contains genetic instructions which direct the microorganism to produce a protein which is encoded by a portion of the heterologous DNA sequence. By growing transformant microorganisms in a fermentor and subjectinq them to conditions under which the heterologous DNA is expressed, valuable proteins can be produced in large quantity at relatively low cost.
Unfortunately, many heterologous proteins which are produced in transformant microorganisms do not fold into their native three-dimensional conformation within the host cell environment. This improper folding of the expressed protein has several untoward consequences. In the first place, the improperly olded proteins tend to form aggregates which are insoluble within the host cell.
These insoluble aggregates are recognizable within the cell as "inclusion bodies", sometimes also referred to as "refractile bodies" and/or "protein granules". The formation of inclusion bodies may also be partially caused by oligomerization of the protein, that is, the Eormation of covalent intermoLecular disulfide bonds.
Not only are the improperly olded proteins insoluble, but also they are biologically inactive. As exemplary of heterologous proteins which form insoluble, biologically ,. '~
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inactive inclusion bodies UpOIl expression in a host cell, one can mention animal growth hormones and growth factors 3uch as bovine growth hormone, porcine growth hormone and somatomedin.
In order to produce useful proteins, it is necessary to convert the improperly folded inclusion body proteins in~o their native con~ormations, in which they are soluble and biologically active. Moreover, it is necessary to purify the protein in order to remove contaminating cell debris and other proteins produced by the host cell. A number oE schemes have been proposed for converting inclusion body proteins into their soluble9 native configurations. Unfortunately, these schemes are often incompatible with protein purification procedures, such as ion-exchange chromatography. The conditions of purification tend to inhibit the ability to maintain the protein in solutionl often resulting in a substantial loss of protein due to reaggregation and precipitation. Most of the schemes proposed for recovering proteins from inclusion bodies in purified, soluble, biologically active Eorm have resulted in very low yields of the proteins produced by the microorganisms.
~J.S. Patent No. 4,511,503 discloses a typical scheme for recovering proteins from inclusion bodies in transformant microorganisms. The inclusion body proteins are treated with a strong denaturant, which causes the improperly folded protein molecules to unfold and become soluble. The denaturant is subsequently removedv for example, by dialysis, in order to allow the protein to refold into its native conformation. The most commonly employed strong denaturant in schemes of this type has been guanidine hydrochloride.
U~S. Patent No. 4,511,502 discloses a similar process wherein the solubilized protein/denaturant ~ 26~ 2,~
solution is passed over a molecular sieve or centrifuged at high speed to remove higher molecular weight components.
U.S. Patent No. 4,518,526 also discloses a similar process. In this process, the transformant cell culture i5 treated with a buffered solution of sufficient ionic strength to solubilize most of the host cell protein, whereas the heterologous protein remains insoluble. The cells are then lysed, the supernatant containing the solubilized host cell protein removed and the insoluble inclusion bodies solubilized in the strong denaturant.
Other publications disclosing denaturation/renaturation schemes for converting inclusion body proteins into their soluble, native conformations include PCT publication WO 83/04418, Æuropean Patent Application Publication No. 0 123 928, European Patent Application Publication No. 0 1~1 775, European Patent Application Publication No. 0 116 778 and European Patent Application Publication No. 0 114 507.
As previously indicated, most of the proposed denaturation/renaturation schemes have employed guanidine hydrochloride as the denaturant. While guanidine hydrochloride is characterized by an excellent ability to solubilize inclusion body proteins, its use entails some problems. Upon dialysis against denaturan~-free buf~er to remove the guanidine hydrochloride, a substantial amount of the solubilized protein reaggregates, apparently due to improper re~olding. ~oreover, when guanidine-solubili~ed protein is puriEied by methods such as ion-exchange chromatography -- which are necessary to obtain the degree of purity required in the final product -- substantial los~es of protein are incurred due to reaggregation. Fouling and plugging of the column tends to occur in an inordinately short time, severely limiting -the useul life o the column. Typically, we have found ~;Z6~
that guanidine solubilization of bovine growth hormone inclu~ion bodies, followed by ion-exchange chromatography, yielded only 4-12% product recovery.
Another major prohlem associated with the use of guanidine hydrochloride -- one which is particularly important from a commercial production standpoint -- is its high cost~ It would be highly desirable to employ a solubilizing agent which is comparable to guanidine hydrochloride in its ability to solubilize inclusion body proteins, but without the associated high cost of guanidine. U.S. Patent No. 4,511,503 suggests the use o detergents such as sodium dodecyl sulfate (SDS) as denaturants. ~owever, there is no demonstration of its use, nor i5 there proposed a method or removing the detergent from the protein and purifying the protein.
Detergents such as SDS are highly effective denaturing agents. Moreover, SDS is a much less expensive reagent than guanidine hydrochloride.
Accordingly, its potential use in recovering inclusion body proteins is attractive from the standpoint of commercial production economics. Compared with guanidine hydrochloride, however, SDS binds to the denatured protein much more tightly, making its complete removal from the protein problematical.
O.H. Kapp and S~W. Vinogradov demonstrated that SDS
could be removed from several proteins by chromatography on the ion-retardation resin AG1lA8 (AnalO Biochem., 91:230-233 ~1978]). It was ~aid that from 0.1 to 1.4 moles of SDS remained on each mole of protein treated in this manner. K. Weber and D.J. Kuter demonstrated that SDS could be removed from aspartate transcarbamylase by incubation in urea and subsequent anion-exchange chromatography (J. Biol. Chem., 246:4504-4509 ~1~71]).
In both instance~, however, the starting proteins were in pure, biologically active form. There is no suggestion ~;,~, , ' . : .
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of a method for e~ficiently recovering protein in a pure, biologically active Eorm from insoluble, intracellular inclusion bodies.
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This invention provides an efficient, economical method for recovering proteins that are produced as insoluble, biologically inactive inclusion body proteins in transformant microorganisms. ~he method of the invention provides for conversion of the protein into its soluble, native conformation and concomitant purification of the protein. In the method o~ the invention, a detergent such as SDS is used as a denaturant, rather than a more expensive denaturant such as guanidine hydrochloride. Quite surprisingly, we have ~ound that losses o~ pr~tein using the method of the invention to purify and activate the protein are substantially less than with the guanidine hydrochloride method. Using SDS
as the denaturant in accordance with the method of the invention, we have obtained recovery yields of up to about 30%, based on the amount of protein present in the inclusion bodies. These yields are significant.l~ higher than those obtainable with prior processes using guanidine hydrochloride as the denaturant. The protein product obtained by the method of ~he invention is soluble, essentially homogeneous and essentially free of SDS.
More particularly, the invention provides a method ~or purifying and activating a protein that is produced as an insoluble, biologically inactive inclusion body in a trans~ormant microorganism which comprises:
(a~ extracting the inclusion bodies into a buffered solution o sodium dodecyl sul~ate to solubilize t~e protein;
(b~ chromatographing the protein solution on an ion-retardation resin to remove the ~26~sJ~
remainder oE the sodium dodecyl sulfate from the protein; and (c) chromatographing the protein solution frorn step (b) on an anion-exchange resin.
In a preferred embodiment of the invention, the protein-containing sodium dodecyl sulfate solution is treated to remove a substantial amount of the sodium dodecyl sulfate prior to chromatography on the ion-1~ retardation resin. Removal of a portion o~ the sodium dodecyl sulfate can be accomplished by dialysis against a buffer solution or by refriyeration of the solution.
DETAIL ~
The method of the invention is used to purify and activate proteins which are produced in the form of insoluble, biologically inactive inclusion bodies in transformant microorganisms, i.e., microorganisms which have been transformed with recombinant DNA vectors that direct the expression of genes coding for heterologous proteins. Generally, the proteins which can be purified and activated by the method of the invention are negatively charged proteins which are basic under the processing conditions that are described below in detail.
Tn speci~ic embodiments o~ the invention, t~e proteins which are purified and activated are animal growth hormones such as bovine growth hormone (bGH~ or porcine growth hormone (pGH).
It is to be understood that re~erence herein to proteins generally -- e.g., hormones and enzymes -- or to specific proteins such as bGH and pGH is not intended to be restricted to molecules which contain the full amino acid sequence of the natural protein. ~ather, it is al90 intended to include fragments of the protein having various portions of t~e sequence deleted and proteins or ~ragments thereof having various substitutions or ..... .. . ~, . .. . , . . .. . .. ~ ~ .. .. . . .. ... . . . .. ... .. . . . . . ..... ..... . . . . ..
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modification~ in their natural sequences (i.e., analogues) which do not destroy the biological activity of the molecules.
The genes for bGH and pGH have been cloned onto expression vectors and used to transform E. coli host cells. European Patent Application Publication No.
0 103 395 describes the construction o~ a transformant strain of E. coli containing a first plasmid which codes for Q9(Ser)b~H (bGH less its 9 N-terminal amino acids and having an additional serine residue at the N-terminus) under the control of the ~PL promoter-operator and which has a Shine-Dalgarno region derived from bacteriophage mu. The transformant also contains a second plasmid, pcI857, which codes ~or the production of the cI857 temperature-sensitive repressor protein. The repressor protein can be inactivated by raising the temperature to about 42C, thereby inducing expression of ~9(Ser)bGH. A trans~ormant strain of this type, E. coli ~B101 (PL-mu-~9(Ser)bG~ and pcI857) has been deposited, with the designation E. coli, IMC No. 1, at The ~nerican Type Culture Collection, Rockville, Maryland, with accession No. 53030.
Co~struction o~ a similar transformant strain which codes for the production of Q7pGH (porcine growth hormone less its first 7 N-terminal amino acids) is described in European Pa~ent Application Publication No. 0 104 920. A
transformant strain of this type, E. coli HB101 ~PL-mu~7pGH and ~cI857) has been deposited, with the de~ignation E~ coli, IMC No. 2, at The ~merican Type Culture Collection, Rockville, Maryland, with accession No. 53031.
E. coli, I~C No. 1 and E. coli, IMC No. 2 are proli~ic producers of Q9(Ser)bGH and Q7pGH, respectively.
In both instances, the expressed protein is sequestered within the cell in the ~orm o~ insoluble, biologically ~126~
inactive inclusion bodies which are visible under a microscope.
After the transformant cells have been grown in a fermentor and the protein of interest has been expressed and allowed to accumulate within the cells as inclusion bodies, the transformant cells are generally lysed, either mechanically, chemically or enzymatically, to allow isolation of the inclusion bodies which are sequestered within the cells~ Prior to employing the method of the invention, the inclusion bodies can be separated from the bulk of the remainder of cellular material by centrifugation and washing in a buffer to produce a wet inclusion body paste.
The inclusion bodies are extracted into a buffered solution of SDS to solubilize the protein. The amount of SDS in the buffered solution is an amount sufficient to dissolve the protein. Preferably, the buffered solution contains from about 0~1% to 500% SDS, most preferably about 1%. We have found that high pH buffers, i.e., from about p~ 805 to p~ 11 are best s-uited to maintaining the protein in a solubiliæed form. A suitable buffer soIution is 0.02 M to ~.1 M ethanolamine-~Cl, carbonate-bicarbonate or borate buffer. If desired, a reducing agent, such as 2-mercaptoethanol, may also be present in the SDS solution in an amount sufficient to prevent the formation of intermolecular disulfide bonds during the recovery proce~s. We have not found, however, that the use of reducing agents results in a signi~icant increase in the yield of soluble, biologically active protein. Disaggregation of the inclusion bodies in the SDS solution generally occurs over a period of about 4 to 18 hours.
Once the inclu~ion body proteins have been solubilized in the SDS solution and allowed to become disaggrega~ed, it is preferred to remove a substantial ~2~JJ~
amount of the SDS from the solution prior to chromatography on the ion-retardation resin. Removal of a substantiaL amount of the SDS can be effected by dialysis against a non SDS-containing buffer solution or by reducing the temperature of the SDS-containing solution. Although it is possible to load the SDS-containing solution directly onto the ion-retardation resin without a preliminary SDS removal step, it is impractical to do so, since removal of all the SDS by means of the ion retardation resin alone would require an impractically large ion-retardation resin column or would entail unacceptably low throughput from a commercial production standpoint. The amount of SDS removed in the preliminary step prior to ion-retardation chromatography t5 can vary, depending primarily on protein concentration.
Typically, Erom about 40% to about 70% of the SDS
initially present in the solution is removed.
As used herein, the term "dialysis" refers to any technique in which SDS is removed from the protein solution by selective transport of SDS ions across a semi-permeable membrane w:ith retention of the desired protein molecules on the other sic1e of the membrane. Any o~ the known methods of dialysis may be used with a ~7ariety of types of equipment. For example, SDS may be dialyzed from the solution using hollow fiber ultrafiltration systems. In these systems, a SDS-free buffer solution of low ionic stren~th is circulated around bundles of semi-permeable hollow fibers. Examples of low ionic strength buffer solutions for use in dialysis include 0.02 to 0.1 M ethanolamine buffer~
carbonate-bicarbonate buffer (HCO3-/CO3-~) or sodium borate buffer. Buffer solutions below about 0.02 M can result in solubility problems, whereas buffers in excess o about 0~1 M ~although useful) are unnecessary. Small molecules in the protein solu~ion that flows through the 1o fibers are c~pable of passing through the membranous fiber wall so as to reduce the ionic strength of the protein solution. Other Xnown dialysis techniques including, but not limited to, diafiltration and sack dialysis can also be employed to remove SDS from the protein solution.
As noted above, refrigeration of the SDS-containing protein solution can be used to remove a portion of the SDS from the protein. The solution containing the solubilized inclusion body protein generally is a low ionic strength buffer solution, such as 0.02 to 0.1 M
carbonate-hicarbonate buffer or borate buffer. A ~ortion of the SDS can be removed from the protein by cooling the buffer solution to a temperature between the freezing point of the ~olution and about 2C for a period o~ about 4 hours, whereupon SDS precipitates out of solution and can be remov~d by filtration or centrifugation.
The dialysis or refrigeration step removes a substantial amount of the SDS from the protein solution.
~owever, some of the SDS will remain tightly bound to the protein. In order to complete the removal of ~SDS from the protein, the protein solution is chromatographed on an ion-retardation resin. An ion-retardation resin is a resin which contains both anion and cation exchange sites and is capable of selectively retarding the flow of ionic substances. It is, therefore, capable of separatin~ the weakly ionic protein molecules from the SDS ions: w~ich pass through the resin more slowly. A pre~erred ion-retardation resin for use in the method of the invention *
is the resin known as AG11A8, which i~ commercially available from Bio-Rad Laboratories, Richmond, California. It is produced by polymerizing acrylic acid inside the resin AG1X8 (a styrene-divinylbenzene copolymer with attached quaternary ammonium groups) and thus contains strongly basic charged cations, ~C~2N-~(CH3)3, and weakly acidic anion3, RCH2C00-.
* Trade Mark .
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The ion-retardation resin is generally employed in the form of a packed column. The column is equilibrated by passing buffer solution (absent protein) through the column until pH and conductivity of feed and eluent match. The protein solution containing SDS is then loaded onto the column. While a variety of eluents can be used to elute the protein from the column, it is pre~erred to elute the column using a bufEered solution of 0.0~ to 0.1 M ethanolamine, preferably about 0.05 M
ethanolamine. The protein, which passes through the column more rapidly than the SDS, is collected in the early-stage eluate. An ~G11A8 column can be regenerated by washing with multiple volumes of 1.0 M NH4Cl, followed by multiple volumes of deionized water or simply by washing with large volumes o~ water.
The eluate from the ion-retardation resin contains SDS-free protein which has been refolded into its native configuration. The eluate from the ion-retardation resin is loaded onto an anion-exchange resin, which is generally in the form of a packed column. One can mention, as merely illustrative of suitable anion-exchange resins, DE~52, DE-53, DEAE-Sepharose CL-68,*Cellufine AM, QAE-~ephadex, Q-Sepharose, QAE-Cellulose and DEAE-Trisacryl. Tl~e anion-exchange resin preferably contains a high degree of substitution with cationic groups, such as quarternary ammoni~n ions, attached to a polysaccharide support, such as dextran, agarose or cellulose, or attached to a polyacrylate support. ~xemplary of such preferred resins is DE-53.
Recently developed synthetic resins which are Xnown to be useful in proteln purification columns, such as Trisacryl ~commercially available from LKB Instruments), can be employed. The protein in the anion-exchange column eluate can be concentrated, i~ desired, using known techniques ~uch as ultrafiltration.
* Trade Mark The purified protein can be eluted from the anion-exchange column using buffered saline solution pre~erably at pH 8.5 or above, with a salt concentration of 0 to 0.1 molar.
After each step in the method of the invention, i.e., after extractio~ into SDS, dialysis or refrigeration, chromatography on the ion-retardation resin and anion-exchange chromatography, the protein-containing solution can be centrifuged, if found desirable or necessary, in order to remove any precipitates which may have formed. The precipitates can either be discarded or recycled.
The following examples are intended to further illustrate the practice of the invention and are not in~ended to limit the ~cope of the invention in any wa~.
EXAMPLE I
Purification and Activation of n ' ~7 Porcine ~rowth ~ormone Inclusion bodies were obtained from E. coli, IMC
No~ 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions. The cells ~rom the fermentor were harvested by centrifugation from the fermentor beer, suspended in 0.1 M phosphate buffer, p~ 7.8, 10 mM ED~A, and lysed by multiple passage through a Manton-Gaulin homogenizer. The crude inclusion bodies were harvested by centrifugation and washed twice in this same buffer.
Twenty-four ~rams of these inclusion bodies were extracted in 1000 ml of 0.1 M ethanolamina-HCl, pH 9.0, 1% SDS, by stirring overnight at room temperature. The resulting extract was dialyzed twice against 16 liters of 20 mM ethanolamine-HCl, p~ 9.0, over a period of 48 hours. Residual insoluble material was removed b~
centrifugation at 15,000 x g Eor 30 minutes. The clarified extract was chromatographed on a 10 x 30 cm :~Z~ 2~
column of AG11A8 ion-retardation resin equilibrated in 50 mM ethanolamine-HCl, pH 9.8. ~he column was developed in this same bu~fer at a linear flow rate of 0.3 cm/min.
The fractions-containing protein ~ere then chromatographed on a 4.4 x 25 cm column of DE-53 cellulose equilibrated in 50 mM ethanolamine-HClt pH 9Ø
After loading the extract, the column was washed with two column volumes of this buffer to elute contaminants in the void volume. NaCl was then added to the buffer to a final concentration of 0.1 M, which caused the elution of the ~7pGH.
The fractions containing pure ~7pGH were concentrated tenfold over an ultrafiltration membrane (10,000 dalton cut-off) under nitrogen pressure, exhaustively dialyzed against 0.~S mM sodium bicarbonate, 0.21 mM sodium carbonate, p~ 9.7, concentrated an additional threefold, and lyophilized. The final product (992 mg) was essentially pure a7pGH. Overall yield, based on the original fermentor titer, was 20.2%.
EXAMPLE II
Purification and Activation of ~7 Porcine Growth Hormone Inclusion bodies were obtaine~ from E. coli, IMC
No. 2 (ATCC 53031), which had been cultured under ~7pG~-producing conditions. The cells were centrifuged out o~ the fermentor beer and resuspended in 0.1 M
Tris-HCl, pH 7.8, 10 mM EDTA, 5% sucrose. Lysozyme was added to 200 mg/liter and incubated at 28C for ~0 minutes, at which time the cells were again centrifuged out. They were resuspended in 0.1 M phosphate buffer, p~
7.8, containing t0 mM EDTA and 0.5 mM reduced glutathione, and lysed by passing twice through a Manton-Gaulin homogenizer at 6000-8000 psi.
Phenylmethylsul~onyl ~luoride wa~ added to 0.5 mM in order to inhibit protease activity and -the crude :L26~
incl~sion bodies were centrifuged out. The pellet was washed -three times by resuspension in 0O1 M Tris-~Cl, pH
7.8, 2 M urea, 1~ Triton X-100, 0.5 mM dithioerythritol ~DTE) followed by centriugation. The remaining pellet was washed twice more with 0.1 ~ Tris-HCl, pH 7.8, 0.5 mM
DTE.
One-hal~ gram of this pellet was extracted in 100 volumes ~50 ml) of "Cornell bufEer minus NaCl" (nCB": 25 mM sodium bicarbonate, 21 mM sodium carbonate, pH 9.8), 1~ w/v SDS, 10 mM DTE, 1 mM EDTA, by stirring for 4 hours under a nitrogen blanket~ This extract was then diluted to 400 ml with CB~10 mM DTE/1 mM EDTA and dialyzed against 10 volumes of CB/5 mM 2-mercaptoethanol (BM~)/1 mM EDTA. The dialyzed extract was chromatographed at a flow rate of 100 ml/hr over a 2.5 x 35 cm column of AG11A8 ion~retardation resin equilibrated in CB/5 mM
BME/l mM EDTA. The protein-containing eluate was then chromatographed at 180 ml/hr flow rate on a 4.4 x 15 cm column of DEAE-Trisacryl equilibrated in this same buffer. A single peak was seen to elute in the void volume of the column and contained >95% pure ~7pGH. The relevant fractions were pooled, concentrated threefold over an ultrafiltration membrane (5000 dalton cu~-off), dialyzed extensively against 1~ C~ (0.25 mM sodium bicarbonate, 0.21 mM sodium carbonate) and lyophilized.
The final product (26.5 mg protein) represented a recovery of 14.5% based on the total amount of protein in the original extract.
EXAMPLE III
Purification and Activation of ~7 Porcine Grc9~e~-c9bs~
.
Inclusion bodies were obtained from E. coli, IMC
No. 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions, in the same manner as was ou~lined in Example I. One gram of these inclusion bodies were extracted in 100 volumes (100 ml) oE CB/1~
SDS/20 mM BME/l mM ~DTA by stirring for 3 hours under a nitrogen blanket. The extract was dialyzed against 2 x 40 volumes of C8/1 mM EDTA and diluted to 400 ml with CB/5 mM BM~/1 mM EDTA. It was then chromatographed on a 4.4 x 30 cm column of AG11A8 resin at 250 ml/hr in CB/5 mM BME/1 mM EDTA. Those fractions containing protein were pooled and chromatographed over a 4.4 x 30 cm column of DEAE-Trisacryl at 150 ml/hr in this same buffer~ When no protein eluted in the void fractions, NaCl was added to a final concentration of 0.2 M, causing a single peak, containing highly purified ~7pGH, to elute The fractions containing pGH were pooled, exhaustively dialyzed against 1~ CB, concentrated 15-fold over an ultrafiltration membrane under 65 psi (5000 dalton cut-off), and lyophilized. The resulting product contained 27 mg of ~7pGH which was >95% pure by gel electrophoretic analysis. The overall yield, based upon total protein content of the orig:inal extract, was 21~.
EXAMPLE IV
Purification and Activation of -~T~ ~~~~Growth Hormone , _ The inclusion bodies used were prep~red from _ coli, IMC No. 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions, in the same manner as was outlined in Example I. Three grams of these inclusions were extracted into 100 volumes of CB/1 A 8%
SDS/20 mM DTE/1 mM EDTA by stirring for 3.5 hours. This extract was then dialy2ed against 2 x 16 volumes of CB/5 mM BME/1 mM EDTA and chromatographed on a 4.4 x 24 cm AG1lA8 column equilibrated in the same bu~fer. The column was developed with this buffer at a linear flow rate of 0.3 cm/min. The fractions containin~ protein were pooled and extensively dialyzed against 50 ~M
~26~,~4~
ethanolamine-HCl, p~ 9.0, and chromatographed on a 4.4 x 20 cm column of DE-53 in the same bufEer. No protein eluted in the brea~through fractions, but addition of 0.1 M NaCl to the buffer caused elution of the ~7pGH. The relevant fractions were pooled, diafiltered in a stirred cell over a 5000-dalton cut-off membrane against 5 mM
Tris-HCl, pH 7.4, concentrated roughly tenfold, and lyophilized. The final product was 29 mg of powder that was >90~ pure ~7pGH by gel analysis. Overall yield, based on the original ~7pGH content of the inclusion bodies, was 5.8%.
EXAMPLE V
Purification and Activation of ~9_Ser)bG8 Inclusion bodies were obtained from E. coli, IMC
No. 1 (ATCC 53030), which had been cultured under ~9(Ser~bGH-producing conditions. Live cells harvested from the fermentor beer (830.6 g cell paste) were temporarily stored at -8SC, then thawed, treated with lysozyme, homogenized, centrifuged and resuspended in 0.1 M phosphate buffer, pH 7.8, 10 mM EDT~. The cells were then lysed by three passages through a Manton-Gaulin homogenizer at 6000 psi. Following centrifugation (14,000 x g, 20 min.), 357.5 g of inclusions were recovered. These were resuspended in 500 ml 100 mM
Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 0.5 mM PMSF and stored at -85C. About one-third of this material t131~5 g) was thawed and used in the purification/activation process. The crude inclusions were washed once with 10 volumes of 0.2 M Na~2PO4, 10 mM EDTA, pH 7.8, and centrifuged to yield 109.3 g of pellet. The washed inclusions were solubilized by 60 sec. homogeni2ation into 6,560 ml of 0.1 M ethanolamine-~Cl, pH ~.0, 1% SDS.
After overnight stirring, this extract was diafiltered against ten volumes of 20 mM ethanolamine-~Cl, pH 9.0, 3S which lowered the SDS level to 4.8 mg/ml. The extract ~l~6~;J~.,i9~
was centrifuged at 8000 rpm for 30 ~inutes to remove particulates, diluted 1:1 in 20 mM ethanolamine-HCl, p~
9.0, and chromatographed on a 15-liter AG1lA8 column.
The ~(Ser)bGH pool from the AG1lA8 column was then chromatographed on a 1.85-liter ~E 52 column equilibrated in 50 mM ethanolamine-HCl, pM 9.0 at 16 ml/min. There was a considerable amount of protein in the breakthrough fraction as indicated by the A280. This plus a one column-volume wash with 50 mM ethanolamine-HCl, pH 9.0, constituted 18 liters of material~ Since eluate was still exhibiting non-zero A280, an additional 8 liters of material was collected. Approximately 2 column-volume washes each of 0.1 and 1.0 M NaCl followed; those were collected separately. The breakthrough fractions contained >95~ pure Q9(Ser)bGH by SDS-PAGE analysis.
Protein determination by Coomassie Blue dye binding assay showed ~3.2 g of protein in the pooled eluate. The NaCl washes also contained ~9lSer)bG~, but it was almost completely covalently aggregat~d. The 18 liters of DE-52 eluant from the DE-52 column were concentrated 4.5-fold in an Amicon DC2 ultrafiltration unit, after which air injected during recirculation caused ~rothing and precipitation.
The precipitate was re~oved by filtration thr~ugh Whatman No. 2 filter paper and final concentration to about 2 liter~ was accomplished over a YM-10 membrane.
~he protein solution was then dialyzed (sack dialysis~
three times against 36 liters o~ 1~ Cornell bufer minus NaCl and lyophilized to yield 2.8 grams o~ product.
The 8 liters of additional material washed off the DE-52 column was analyzed and appeared to constitute essentially pure Q9(Ser)bGH. A~ter concentration to 1.3 liters in a stirred cell, Bradford assays indicated the presence of 1.25 g o~ protein.
Based upon the original titer in the ~ermentort the combined Q9(Ser)bG~ recovered in pure, soluble form in ~26'~
the 18-liter breakthrough ~raction and the 8 liter wash from the DE-52 column represents a combined yield on the order of about 30%.
EXAMPLE VI
Purification and Activation of Q~(Ser)bGH
Inclusion bodies obtained from E. coli, IMC No. 1 (ATCC 53030) are extracted in 100 volumes of CB
containing 1% SDS by stirring overnight. The resulting extract is refrigerated at O~C for 4 hours. Precipitated SDS and residual insoluble inclusion body material are removed by centrifugation. The clarified extract, a~ter coming to room temperature, is chromatographed over a packed AG1lA8 column equilibrated with CB at a linear flow rate of 0.3 cm/min. The fractions containing protein are pooled and loaded onto a column of DEAE Sepharose Fast Flow which has been equilihrated in CB, the pH of which has been adjusted to 9.0 with concentrated HCl. The column is developed with CB
containing 0.1 M NaCl~ The ~9(Ser)bGH-containing fractions are pooled, concentrated roughly 5-fold over a PM-10 ultrafiltration membrane, dialyzed into 5 mM
Tris-HCl, pH 7.4, and lyophilized to yield the final product.
EXAMPLE VII
Determination of Residual SDS in Purified, ~ G~1r~D~5~ ~~eins _ Each of the final protein solutions obtained in Bxamples I through IV was analyzed to determine the amount of residual SDS, if any, which was present. The SDS content of the final product was determined by preparing a 10 mg/ml solution in CB. A 100-~l aliquot of this solution wa~ mixed with an equal volume o~ 1~
acridine orange in 0.5 M NaH.S04. Three ml o~ toluene were added and the tube capped and shaken vigorously for 3 minutes. The aqueous and organic phases were separated ,:, ~a~6~
by centrifugation for 5 minutes in a clinical centrifuge.
The top ttoluene) layer was removed to a glass cuvette and its absorbance at 499 nm determined. The SDS level was determined from a standard curve constructed by performing this operation on a series of SDS solutions of known concentration (range 0-150 ~g/ml). In each instance, the SDS level was below the quantitative lower limit o the assay (around 10 ~g/ml).
~AC K r R l~tJND OF THE INVEETION
This invention relates to methods for puriEying and activating proteins that are produced as insoluble, biologically inacti~e inclusion bodies in microorganisms that have been transformed with recombinant DNA
expression vectors to direct expression of the protein of interest.
Recombinant DNA technology allows the insertion of a vector carrying foreign (heterologous) DNA into a microorganism in a manner which allows the heterologous DNA to be expressed; that is, the vector contains genetic instructions which direct the microorganism to produce a protein which is encoded by a portion of the heterologous DNA sequence. By growing transformant microorganisms in a fermentor and subjectinq them to conditions under which the heterologous DNA is expressed, valuable proteins can be produced in large quantity at relatively low cost.
Unfortunately, many heterologous proteins which are produced in transformant microorganisms do not fold into their native three-dimensional conformation within the host cell environment. This improper folding of the expressed protein has several untoward consequences. In the first place, the improperly olded proteins tend to form aggregates which are insoluble within the host cell.
These insoluble aggregates are recognizable within the cell as "inclusion bodies", sometimes also referred to as "refractile bodies" and/or "protein granules". The formation of inclusion bodies may also be partially caused by oligomerization of the protein, that is, the Eormation of covalent intermoLecular disulfide bonds.
Not only are the improperly olded proteins insoluble, but also they are biologically inactive. As exemplary of heterologous proteins which form insoluble, biologically ,. '~
, . , . . .... . . ~ ,.. . .. . .
~6'iJ~
inactive inclusion bodies UpOIl expression in a host cell, one can mention animal growth hormones and growth factors 3uch as bovine growth hormone, porcine growth hormone and somatomedin.
In order to produce useful proteins, it is necessary to convert the improperly folded inclusion body proteins in~o their native con~ormations, in which they are soluble and biologically active. Moreover, it is necessary to purify the protein in order to remove contaminating cell debris and other proteins produced by the host cell. A number oE schemes have been proposed for converting inclusion body proteins into their soluble9 native configurations. Unfortunately, these schemes are often incompatible with protein purification procedures, such as ion-exchange chromatography. The conditions of purification tend to inhibit the ability to maintain the protein in solutionl often resulting in a substantial loss of protein due to reaggregation and precipitation. Most of the schemes proposed for recovering proteins from inclusion bodies in purified, soluble, biologically active Eorm have resulted in very low yields of the proteins produced by the microorganisms.
~J.S. Patent No. 4,511,503 discloses a typical scheme for recovering proteins from inclusion bodies in transformant microorganisms. The inclusion body proteins are treated with a strong denaturant, which causes the improperly folded protein molecules to unfold and become soluble. The denaturant is subsequently removedv for example, by dialysis, in order to allow the protein to refold into its native conformation. The most commonly employed strong denaturant in schemes of this type has been guanidine hydrochloride.
U~S. Patent No. 4,511,502 discloses a similar process wherein the solubilized protein/denaturant ~ 26~ 2,~
solution is passed over a molecular sieve or centrifuged at high speed to remove higher molecular weight components.
U.S. Patent No. 4,518,526 also discloses a similar process. In this process, the transformant cell culture i5 treated with a buffered solution of sufficient ionic strength to solubilize most of the host cell protein, whereas the heterologous protein remains insoluble. The cells are then lysed, the supernatant containing the solubilized host cell protein removed and the insoluble inclusion bodies solubilized in the strong denaturant.
Other publications disclosing denaturation/renaturation schemes for converting inclusion body proteins into their soluble, native conformations include PCT publication WO 83/04418, Æuropean Patent Application Publication No. 0 123 928, European Patent Application Publication No. 0 1~1 775, European Patent Application Publication No. 0 116 778 and European Patent Application Publication No. 0 114 507.
As previously indicated, most of the proposed denaturation/renaturation schemes have employed guanidine hydrochloride as the denaturant. While guanidine hydrochloride is characterized by an excellent ability to solubilize inclusion body proteins, its use entails some problems. Upon dialysis against denaturan~-free buf~er to remove the guanidine hydrochloride, a substantial amount of the solubilized protein reaggregates, apparently due to improper re~olding. ~oreover, when guanidine-solubili~ed protein is puriEied by methods such as ion-exchange chromatography -- which are necessary to obtain the degree of purity required in the final product -- substantial los~es of protein are incurred due to reaggregation. Fouling and plugging of the column tends to occur in an inordinately short time, severely limiting -the useul life o the column. Typically, we have found ~;Z6~
that guanidine solubilization of bovine growth hormone inclu~ion bodies, followed by ion-exchange chromatography, yielded only 4-12% product recovery.
Another major prohlem associated with the use of guanidine hydrochloride -- one which is particularly important from a commercial production standpoint -- is its high cost~ It would be highly desirable to employ a solubilizing agent which is comparable to guanidine hydrochloride in its ability to solubilize inclusion body proteins, but without the associated high cost of guanidine. U.S. Patent No. 4,511,503 suggests the use o detergents such as sodium dodecyl sulfate (SDS) as denaturants. ~owever, there is no demonstration of its use, nor i5 there proposed a method or removing the detergent from the protein and purifying the protein.
Detergents such as SDS are highly effective denaturing agents. Moreover, SDS is a much less expensive reagent than guanidine hydrochloride.
Accordingly, its potential use in recovering inclusion body proteins is attractive from the standpoint of commercial production economics. Compared with guanidine hydrochloride, however, SDS binds to the denatured protein much more tightly, making its complete removal from the protein problematical.
O.H. Kapp and S~W. Vinogradov demonstrated that SDS
could be removed from several proteins by chromatography on the ion-retardation resin AG1lA8 (AnalO Biochem., 91:230-233 ~1978]). It was ~aid that from 0.1 to 1.4 moles of SDS remained on each mole of protein treated in this manner. K. Weber and D.J. Kuter demonstrated that SDS could be removed from aspartate transcarbamylase by incubation in urea and subsequent anion-exchange chromatography (J. Biol. Chem., 246:4504-4509 ~1~71]).
In both instance~, however, the starting proteins were in pure, biologically active form. There is no suggestion ~;,~, , ' . : .
:~.2~4~
of a method for e~ficiently recovering protein in a pure, biologically active Eorm from insoluble, intracellular inclusion bodies.
~U~
This invention provides an efficient, economical method for recovering proteins that are produced as insoluble, biologically inactive inclusion body proteins in transformant microorganisms. ~he method of the invention provides for conversion of the protein into its soluble, native conformation and concomitant purification of the protein. In the method o~ the invention, a detergent such as SDS is used as a denaturant, rather than a more expensive denaturant such as guanidine hydrochloride. Quite surprisingly, we have ~ound that losses o~ pr~tein using the method of the invention to purify and activate the protein are substantially less than with the guanidine hydrochloride method. Using SDS
as the denaturant in accordance with the method of the invention, we have obtained recovery yields of up to about 30%, based on the amount of protein present in the inclusion bodies. These yields are significant.l~ higher than those obtainable with prior processes using guanidine hydrochloride as the denaturant. The protein product obtained by the method of ~he invention is soluble, essentially homogeneous and essentially free of SDS.
More particularly, the invention provides a method ~or purifying and activating a protein that is produced as an insoluble, biologically inactive inclusion body in a trans~ormant microorganism which comprises:
(a~ extracting the inclusion bodies into a buffered solution o sodium dodecyl sul~ate to solubilize t~e protein;
(b~ chromatographing the protein solution on an ion-retardation resin to remove the ~26~sJ~
remainder oE the sodium dodecyl sulfate from the protein; and (c) chromatographing the protein solution frorn step (b) on an anion-exchange resin.
In a preferred embodiment of the invention, the protein-containing sodium dodecyl sulfate solution is treated to remove a substantial amount of the sodium dodecyl sulfate prior to chromatography on the ion-1~ retardation resin. Removal of a portion o~ the sodium dodecyl sulfate can be accomplished by dialysis against a buffer solution or by refriyeration of the solution.
DETAIL ~
The method of the invention is used to purify and activate proteins which are produced in the form of insoluble, biologically inactive inclusion bodies in transformant microorganisms, i.e., microorganisms which have been transformed with recombinant DNA vectors that direct the expression of genes coding for heterologous proteins. Generally, the proteins which can be purified and activated by the method of the invention are negatively charged proteins which are basic under the processing conditions that are described below in detail.
Tn speci~ic embodiments o~ the invention, t~e proteins which are purified and activated are animal growth hormones such as bovine growth hormone (bGH~ or porcine growth hormone (pGH).
It is to be understood that re~erence herein to proteins generally -- e.g., hormones and enzymes -- or to specific proteins such as bGH and pGH is not intended to be restricted to molecules which contain the full amino acid sequence of the natural protein. ~ather, it is al90 intended to include fragments of the protein having various portions of t~e sequence deleted and proteins or ~ragments thereof having various substitutions or ..... .. . ~, . .. . , . . .. . .. ~ ~ .. .. . . .. ... . . . .. ... .. . . . . . ..... ..... . . . . ..
. .
, ~7~
modification~ in their natural sequences (i.e., analogues) which do not destroy the biological activity of the molecules.
The genes for bGH and pGH have been cloned onto expression vectors and used to transform E. coli host cells. European Patent Application Publication No.
0 103 395 describes the construction o~ a transformant strain of E. coli containing a first plasmid which codes for Q9(Ser)b~H (bGH less its 9 N-terminal amino acids and having an additional serine residue at the N-terminus) under the control of the ~PL promoter-operator and which has a Shine-Dalgarno region derived from bacteriophage mu. The transformant also contains a second plasmid, pcI857, which codes ~or the production of the cI857 temperature-sensitive repressor protein. The repressor protein can be inactivated by raising the temperature to about 42C, thereby inducing expression of ~9(Ser)bGH. A trans~ormant strain of this type, E. coli ~B101 (PL-mu-~9(Ser)bG~ and pcI857) has been deposited, with the designation E. coli, IMC No. 1, at The ~nerican Type Culture Collection, Rockville, Maryland, with accession No. 53030.
Co~struction o~ a similar transformant strain which codes for the production of Q7pGH (porcine growth hormone less its first 7 N-terminal amino acids) is described in European Pa~ent Application Publication No. 0 104 920. A
transformant strain of this type, E. coli HB101 ~PL-mu~7pGH and ~cI857) has been deposited, with the de~ignation E~ coli, IMC No. 2, at The ~merican Type Culture Collection, Rockville, Maryland, with accession No. 53031.
E. coli, I~C No. 1 and E. coli, IMC No. 2 are proli~ic producers of Q9(Ser)bGH and Q7pGH, respectively.
In both instances, the expressed protein is sequestered within the cell in the ~orm o~ insoluble, biologically ~126~
inactive inclusion bodies which are visible under a microscope.
After the transformant cells have been grown in a fermentor and the protein of interest has been expressed and allowed to accumulate within the cells as inclusion bodies, the transformant cells are generally lysed, either mechanically, chemically or enzymatically, to allow isolation of the inclusion bodies which are sequestered within the cells~ Prior to employing the method of the invention, the inclusion bodies can be separated from the bulk of the remainder of cellular material by centrifugation and washing in a buffer to produce a wet inclusion body paste.
The inclusion bodies are extracted into a buffered solution of SDS to solubilize the protein. The amount of SDS in the buffered solution is an amount sufficient to dissolve the protein. Preferably, the buffered solution contains from about 0~1% to 500% SDS, most preferably about 1%. We have found that high pH buffers, i.e., from about p~ 805 to p~ 11 are best s-uited to maintaining the protein in a solubiliæed form. A suitable buffer soIution is 0.02 M to ~.1 M ethanolamine-~Cl, carbonate-bicarbonate or borate buffer. If desired, a reducing agent, such as 2-mercaptoethanol, may also be present in the SDS solution in an amount sufficient to prevent the formation of intermolecular disulfide bonds during the recovery proce~s. We have not found, however, that the use of reducing agents results in a signi~icant increase in the yield of soluble, biologically active protein. Disaggregation of the inclusion bodies in the SDS solution generally occurs over a period of about 4 to 18 hours.
Once the inclu~ion body proteins have been solubilized in the SDS solution and allowed to become disaggrega~ed, it is preferred to remove a substantial ~2~JJ~
amount of the SDS from the solution prior to chromatography on the ion-retardation resin. Removal of a substantiaL amount of the SDS can be effected by dialysis against a non SDS-containing buffer solution or by reducing the temperature of the SDS-containing solution. Although it is possible to load the SDS-containing solution directly onto the ion-retardation resin without a preliminary SDS removal step, it is impractical to do so, since removal of all the SDS by means of the ion retardation resin alone would require an impractically large ion-retardation resin column or would entail unacceptably low throughput from a commercial production standpoint. The amount of SDS removed in the preliminary step prior to ion-retardation chromatography t5 can vary, depending primarily on protein concentration.
Typically, Erom about 40% to about 70% of the SDS
initially present in the solution is removed.
As used herein, the term "dialysis" refers to any technique in which SDS is removed from the protein solution by selective transport of SDS ions across a semi-permeable membrane w:ith retention of the desired protein molecules on the other sic1e of the membrane. Any o~ the known methods of dialysis may be used with a ~7ariety of types of equipment. For example, SDS may be dialyzed from the solution using hollow fiber ultrafiltration systems. In these systems, a SDS-free buffer solution of low ionic stren~th is circulated around bundles of semi-permeable hollow fibers. Examples of low ionic strength buffer solutions for use in dialysis include 0.02 to 0.1 M ethanolamine buffer~
carbonate-bicarbonate buffer (HCO3-/CO3-~) or sodium borate buffer. Buffer solutions below about 0.02 M can result in solubility problems, whereas buffers in excess o about 0~1 M ~although useful) are unnecessary. Small molecules in the protein solu~ion that flows through the 1o fibers are c~pable of passing through the membranous fiber wall so as to reduce the ionic strength of the protein solution. Other Xnown dialysis techniques including, but not limited to, diafiltration and sack dialysis can also be employed to remove SDS from the protein solution.
As noted above, refrigeration of the SDS-containing protein solution can be used to remove a portion of the SDS from the protein. The solution containing the solubilized inclusion body protein generally is a low ionic strength buffer solution, such as 0.02 to 0.1 M
carbonate-hicarbonate buffer or borate buffer. A ~ortion of the SDS can be removed from the protein by cooling the buffer solution to a temperature between the freezing point of the ~olution and about 2C for a period o~ about 4 hours, whereupon SDS precipitates out of solution and can be remov~d by filtration or centrifugation.
The dialysis or refrigeration step removes a substantial amount of the SDS from the protein solution.
~owever, some of the SDS will remain tightly bound to the protein. In order to complete the removal of ~SDS from the protein, the protein solution is chromatographed on an ion-retardation resin. An ion-retardation resin is a resin which contains both anion and cation exchange sites and is capable of selectively retarding the flow of ionic substances. It is, therefore, capable of separatin~ the weakly ionic protein molecules from the SDS ions: w~ich pass through the resin more slowly. A pre~erred ion-retardation resin for use in the method of the invention *
is the resin known as AG11A8, which i~ commercially available from Bio-Rad Laboratories, Richmond, California. It is produced by polymerizing acrylic acid inside the resin AG1X8 (a styrene-divinylbenzene copolymer with attached quaternary ammonium groups) and thus contains strongly basic charged cations, ~C~2N-~(CH3)3, and weakly acidic anion3, RCH2C00-.
* Trade Mark .
-~6~JJ~
The ion-retardation resin is generally employed in the form of a packed column. The column is equilibrated by passing buffer solution (absent protein) through the column until pH and conductivity of feed and eluent match. The protein solution containing SDS is then loaded onto the column. While a variety of eluents can be used to elute the protein from the column, it is pre~erred to elute the column using a bufEered solution of 0.0~ to 0.1 M ethanolamine, preferably about 0.05 M
ethanolamine. The protein, which passes through the column more rapidly than the SDS, is collected in the early-stage eluate. An ~G11A8 column can be regenerated by washing with multiple volumes of 1.0 M NH4Cl, followed by multiple volumes of deionized water or simply by washing with large volumes o~ water.
The eluate from the ion-retardation resin contains SDS-free protein which has been refolded into its native configuration. The eluate from the ion-retardation resin is loaded onto an anion-exchange resin, which is generally in the form of a packed column. One can mention, as merely illustrative of suitable anion-exchange resins, DE~52, DE-53, DEAE-Sepharose CL-68,*Cellufine AM, QAE-~ephadex, Q-Sepharose, QAE-Cellulose and DEAE-Trisacryl. Tl~e anion-exchange resin preferably contains a high degree of substitution with cationic groups, such as quarternary ammoni~n ions, attached to a polysaccharide support, such as dextran, agarose or cellulose, or attached to a polyacrylate support. ~xemplary of such preferred resins is DE-53.
Recently developed synthetic resins which are Xnown to be useful in proteln purification columns, such as Trisacryl ~commercially available from LKB Instruments), can be employed. The protein in the anion-exchange column eluate can be concentrated, i~ desired, using known techniques ~uch as ultrafiltration.
* Trade Mark The purified protein can be eluted from the anion-exchange column using buffered saline solution pre~erably at pH 8.5 or above, with a salt concentration of 0 to 0.1 molar.
After each step in the method of the invention, i.e., after extractio~ into SDS, dialysis or refrigeration, chromatography on the ion-retardation resin and anion-exchange chromatography, the protein-containing solution can be centrifuged, if found desirable or necessary, in order to remove any precipitates which may have formed. The precipitates can either be discarded or recycled.
The following examples are intended to further illustrate the practice of the invention and are not in~ended to limit the ~cope of the invention in any wa~.
EXAMPLE I
Purification and Activation of n ' ~7 Porcine ~rowth ~ormone Inclusion bodies were obtained from E. coli, IMC
No~ 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions. The cells ~rom the fermentor were harvested by centrifugation from the fermentor beer, suspended in 0.1 M phosphate buffer, p~ 7.8, 10 mM ED~A, and lysed by multiple passage through a Manton-Gaulin homogenizer. The crude inclusion bodies were harvested by centrifugation and washed twice in this same buffer.
Twenty-four ~rams of these inclusion bodies were extracted in 1000 ml of 0.1 M ethanolamina-HCl, pH 9.0, 1% SDS, by stirring overnight at room temperature. The resulting extract was dialyzed twice against 16 liters of 20 mM ethanolamine-HCl, p~ 9.0, over a period of 48 hours. Residual insoluble material was removed b~
centrifugation at 15,000 x g Eor 30 minutes. The clarified extract was chromatographed on a 10 x 30 cm :~Z~ 2~
column of AG11A8 ion-retardation resin equilibrated in 50 mM ethanolamine-HCl, pH 9.8. ~he column was developed in this same bu~fer at a linear flow rate of 0.3 cm/min.
The fractions-containing protein ~ere then chromatographed on a 4.4 x 25 cm column of DE-53 cellulose equilibrated in 50 mM ethanolamine-HClt pH 9Ø
After loading the extract, the column was washed with two column volumes of this buffer to elute contaminants in the void volume. NaCl was then added to the buffer to a final concentration of 0.1 M, which caused the elution of the ~7pGH.
The fractions containing pure ~7pGH were concentrated tenfold over an ultrafiltration membrane (10,000 dalton cut-off) under nitrogen pressure, exhaustively dialyzed against 0.~S mM sodium bicarbonate, 0.21 mM sodium carbonate, p~ 9.7, concentrated an additional threefold, and lyophilized. The final product (992 mg) was essentially pure a7pGH. Overall yield, based on the original fermentor titer, was 20.2%.
EXAMPLE II
Purification and Activation of ~7 Porcine Growth Hormone Inclusion bodies were obtaine~ from E. coli, IMC
No. 2 (ATCC 53031), which had been cultured under ~7pG~-producing conditions. The cells were centrifuged out o~ the fermentor beer and resuspended in 0.1 M
Tris-HCl, pH 7.8, 10 mM EDTA, 5% sucrose. Lysozyme was added to 200 mg/liter and incubated at 28C for ~0 minutes, at which time the cells were again centrifuged out. They were resuspended in 0.1 M phosphate buffer, p~
7.8, containing t0 mM EDTA and 0.5 mM reduced glutathione, and lysed by passing twice through a Manton-Gaulin homogenizer at 6000-8000 psi.
Phenylmethylsul~onyl ~luoride wa~ added to 0.5 mM in order to inhibit protease activity and -the crude :L26~
incl~sion bodies were centrifuged out. The pellet was washed -three times by resuspension in 0O1 M Tris-~Cl, pH
7.8, 2 M urea, 1~ Triton X-100, 0.5 mM dithioerythritol ~DTE) followed by centriugation. The remaining pellet was washed twice more with 0.1 ~ Tris-HCl, pH 7.8, 0.5 mM
DTE.
One-hal~ gram of this pellet was extracted in 100 volumes ~50 ml) of "Cornell bufEer minus NaCl" (nCB": 25 mM sodium bicarbonate, 21 mM sodium carbonate, pH 9.8), 1~ w/v SDS, 10 mM DTE, 1 mM EDTA, by stirring for 4 hours under a nitrogen blanket~ This extract was then diluted to 400 ml with CB~10 mM DTE/1 mM EDTA and dialyzed against 10 volumes of CB/5 mM 2-mercaptoethanol (BM~)/1 mM EDTA. The dialyzed extract was chromatographed at a flow rate of 100 ml/hr over a 2.5 x 35 cm column of AG11A8 ion~retardation resin equilibrated in CB/5 mM
BME/l mM EDTA. The protein-containing eluate was then chromatographed at 180 ml/hr flow rate on a 4.4 x 15 cm column of DEAE-Trisacryl equilibrated in this same buffer. A single peak was seen to elute in the void volume of the column and contained >95% pure ~7pGH. The relevant fractions were pooled, concentrated threefold over an ultrafiltration membrane (5000 dalton cu~-off), dialyzed extensively against 1~ C~ (0.25 mM sodium bicarbonate, 0.21 mM sodium carbonate) and lyophilized.
The final product (26.5 mg protein) represented a recovery of 14.5% based on the total amount of protein in the original extract.
EXAMPLE III
Purification and Activation of ~7 Porcine Grc9~e~-c9bs~
.
Inclusion bodies were obtained from E. coli, IMC
No. 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions, in the same manner as was ou~lined in Example I. One gram of these inclusion bodies were extracted in 100 volumes (100 ml) oE CB/1~
SDS/20 mM BME/l mM ~DTA by stirring for 3 hours under a nitrogen blanket. The extract was dialyzed against 2 x 40 volumes of C8/1 mM EDTA and diluted to 400 ml with CB/5 mM BM~/1 mM EDTA. It was then chromatographed on a 4.4 x 30 cm column of AG11A8 resin at 250 ml/hr in CB/5 mM BME/1 mM EDTA. Those fractions containing protein were pooled and chromatographed over a 4.4 x 30 cm column of DEAE-Trisacryl at 150 ml/hr in this same buffer~ When no protein eluted in the void fractions, NaCl was added to a final concentration of 0.2 M, causing a single peak, containing highly purified ~7pGH, to elute The fractions containing pGH were pooled, exhaustively dialyzed against 1~ CB, concentrated 15-fold over an ultrafiltration membrane under 65 psi (5000 dalton cut-off), and lyophilized. The resulting product contained 27 mg of ~7pGH which was >95% pure by gel electrophoretic analysis. The overall yield, based upon total protein content of the orig:inal extract, was 21~.
EXAMPLE IV
Purification and Activation of -~T~ ~~~~Growth Hormone , _ The inclusion bodies used were prep~red from _ coli, IMC No. 2 (ATCC 53031), which had been cultured under ~7pGH-producing conditions, in the same manner as was outlined in Example I. Three grams of these inclusions were extracted into 100 volumes of CB/1 A 8%
SDS/20 mM DTE/1 mM EDTA by stirring for 3.5 hours. This extract was then dialy2ed against 2 x 16 volumes of CB/5 mM BME/1 mM EDTA and chromatographed on a 4.4 x 24 cm AG1lA8 column equilibrated in the same bu~fer. The column was developed with this buffer at a linear flow rate of 0.3 cm/min. The fractions containin~ protein were pooled and extensively dialyzed against 50 ~M
~26~,~4~
ethanolamine-HCl, p~ 9.0, and chromatographed on a 4.4 x 20 cm column of DE-53 in the same bufEer. No protein eluted in the brea~through fractions, but addition of 0.1 M NaCl to the buffer caused elution of the ~7pGH. The relevant fractions were pooled, diafiltered in a stirred cell over a 5000-dalton cut-off membrane against 5 mM
Tris-HCl, pH 7.4, concentrated roughly tenfold, and lyophilized. The final product was 29 mg of powder that was >90~ pure ~7pGH by gel analysis. Overall yield, based on the original ~7pGH content of the inclusion bodies, was 5.8%.
EXAMPLE V
Purification and Activation of ~9_Ser)bG8 Inclusion bodies were obtained from E. coli, IMC
No. 1 (ATCC 53030), which had been cultured under ~9(Ser~bGH-producing conditions. Live cells harvested from the fermentor beer (830.6 g cell paste) were temporarily stored at -8SC, then thawed, treated with lysozyme, homogenized, centrifuged and resuspended in 0.1 M phosphate buffer, pH 7.8, 10 mM EDT~. The cells were then lysed by three passages through a Manton-Gaulin homogenizer at 6000 psi. Following centrifugation (14,000 x g, 20 min.), 357.5 g of inclusions were recovered. These were resuspended in 500 ml 100 mM
Tris-HCl, pH 7.5, 0.5 mM dithiothreitol, 0.5 mM PMSF and stored at -85C. About one-third of this material t131~5 g) was thawed and used in the purification/activation process. The crude inclusions were washed once with 10 volumes of 0.2 M Na~2PO4, 10 mM EDTA, pH 7.8, and centrifuged to yield 109.3 g of pellet. The washed inclusions were solubilized by 60 sec. homogeni2ation into 6,560 ml of 0.1 M ethanolamine-~Cl, pH ~.0, 1% SDS.
After overnight stirring, this extract was diafiltered against ten volumes of 20 mM ethanolamine-~Cl, pH 9.0, 3S which lowered the SDS level to 4.8 mg/ml. The extract ~l~6~;J~.,i9~
was centrifuged at 8000 rpm for 30 ~inutes to remove particulates, diluted 1:1 in 20 mM ethanolamine-HCl, p~
9.0, and chromatographed on a 15-liter AG1lA8 column.
The ~(Ser)bGH pool from the AG1lA8 column was then chromatographed on a 1.85-liter ~E 52 column equilibrated in 50 mM ethanolamine-HCl, pM 9.0 at 16 ml/min. There was a considerable amount of protein in the breakthrough fraction as indicated by the A280. This plus a one column-volume wash with 50 mM ethanolamine-HCl, pH 9.0, constituted 18 liters of material~ Since eluate was still exhibiting non-zero A280, an additional 8 liters of material was collected. Approximately 2 column-volume washes each of 0.1 and 1.0 M NaCl followed; those were collected separately. The breakthrough fractions contained >95~ pure Q9(Ser)bGH by SDS-PAGE analysis.
Protein determination by Coomassie Blue dye binding assay showed ~3.2 g of protein in the pooled eluate. The NaCl washes also contained ~9lSer)bG~, but it was almost completely covalently aggregat~d. The 18 liters of DE-52 eluant from the DE-52 column were concentrated 4.5-fold in an Amicon DC2 ultrafiltration unit, after which air injected during recirculation caused ~rothing and precipitation.
The precipitate was re~oved by filtration thr~ugh Whatman No. 2 filter paper and final concentration to about 2 liter~ was accomplished over a YM-10 membrane.
~he protein solution was then dialyzed (sack dialysis~
three times against 36 liters o~ 1~ Cornell bufer minus NaCl and lyophilized to yield 2.8 grams o~ product.
The 8 liters of additional material washed off the DE-52 column was analyzed and appeared to constitute essentially pure Q9(Ser)bGH. A~ter concentration to 1.3 liters in a stirred cell, Bradford assays indicated the presence of 1.25 g o~ protein.
Based upon the original titer in the ~ermentort the combined Q9(Ser)bG~ recovered in pure, soluble form in ~26'~
the 18-liter breakthrough ~raction and the 8 liter wash from the DE-52 column represents a combined yield on the order of about 30%.
EXAMPLE VI
Purification and Activation of Q~(Ser)bGH
Inclusion bodies obtained from E. coli, IMC No. 1 (ATCC 53030) are extracted in 100 volumes of CB
containing 1% SDS by stirring overnight. The resulting extract is refrigerated at O~C for 4 hours. Precipitated SDS and residual insoluble inclusion body material are removed by centrifugation. The clarified extract, a~ter coming to room temperature, is chromatographed over a packed AG1lA8 column equilibrated with CB at a linear flow rate of 0.3 cm/min. The fractions containing protein are pooled and loaded onto a column of DEAE Sepharose Fast Flow which has been equilihrated in CB, the pH of which has been adjusted to 9.0 with concentrated HCl. The column is developed with CB
containing 0.1 M NaCl~ The ~9(Ser)bGH-containing fractions are pooled, concentrated roughly 5-fold over a PM-10 ultrafiltration membrane, dialyzed into 5 mM
Tris-HCl, pH 7.4, and lyophilized to yield the final product.
EXAMPLE VII
Determination of Residual SDS in Purified, ~ G~1r~D~5~ ~~eins _ Each of the final protein solutions obtained in Bxamples I through IV was analyzed to determine the amount of residual SDS, if any, which was present. The SDS content of the final product was determined by preparing a 10 mg/ml solution in CB. A 100-~l aliquot of this solution wa~ mixed with an equal volume o~ 1~
acridine orange in 0.5 M NaH.S04. Three ml o~ toluene were added and the tube capped and shaken vigorously for 3 minutes. The aqueous and organic phases were separated ,:, ~a~6~
by centrifugation for 5 minutes in a clinical centrifuge.
The top ttoluene) layer was removed to a glass cuvette and its absorbance at 499 nm determined. The SDS level was determined from a standard curve constructed by performing this operation on a series of SDS solutions of known concentration (range 0-150 ~g/ml). In each instance, the SDS level was below the quantitative lower limit o the assay (around 10 ~g/ml).
Claims (16)
1. A method for purifying and activating a protein which is produced as an insoluble, biologically inactive inclusion body in a transformant microorganism which comprises:
(a) extracting the inclusion bodies into a buffered solution of sodium dodecyl sulfate to solubilize the protein;
(b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein; and (c) chromatographing the protein solution from step (b) on an anion-exchange resin.
(a) extracting the inclusion bodies into a buffered solution of sodium dodecyl sulfate to solubilize the protein;
(b) chromatographing the protein solution on an ion-retardation resin to remove the sodium dodecyl sulfate from the protein; and (c) chromatographing the protein solution from step (b) on an anion-exchange resin.
2. A method as claimed in claim 1, which further comprises removing a portion of the sodium dodecyl sulfate from the protein solution prior to chromatographing it on the ion-retardation resin.
3. A method as claimed in claim 2, wherein the SDS
is partially removed, prior to ion-retardation chromatography, by dialyzing the protein solution against a SDS-free buffer solution.
is partially removed, prior to ion-retardation chromatography, by dialyzing the protein solution against a SDS-free buffer solution.
4. A method as claimed in claim 3, wherein about 40% to about 70% of the SDS is removed prior to chromatography on the ion-retardation resin.
5. A method as claimed in claim 2, wherein the SDS
is removed, prior to ion-retardation chromatography, by cooling the protein solution to temperature between the freezing point of the solution and about 5°C.
is removed, prior to ion-retardation chromatography, by cooling the protein solution to temperature between the freezing point of the solution and about 5°C.
6. A method as claimed in claim 5, wherein about 40% to about 70% of the SDS is removed prior to chromatography on the ion-retardation resin.
7. A method as claimed in claim 1, wherein the protein is an animal growth hormone.
8. A method as claimed in claim 7, wherein the animal growth hormone is bovine growth hormone or a fragment or analog thereof.
9. A method as claimed in claim 7, wherein the animal growth hormone is porcine growth hormone or a fragment or analog thereof.
10. A method as claimed in claim 1, wherein the inclusion bodies are extracted into a buffered solution containing from about 0.1% to 5.0% sodium dodecyl sulfate.
11. A method as claimed in claim 1, wherein the inclusion bodies are extracted into a buffered solution containing about 1% sodium dodecyl sulfate.
12. A method as claimed in claim 1, wherein the sodium dodecyl sulfate solution has a pH from about pH
8.5 to pH 11.
8.5 to pH 11.
13. A method as claimed in claim 1, wherein the ion-retardation resin is AG11A8.
14. A method as claimed in claim 1, wherein the anion-exchange resin is DE-53.
15. A method as claimed in claim 8, wherein the protein is .DELTA.9(Ser)bGH.
16. A method as claimed in claim 9, wherein the protein is .DELTA.7 pGH.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000517604A CA1267496A (en) | 1985-09-06 | 1986-09-05 | Purification and activation of proteins from insoluble inclusion bodies |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US06/773,174 US4677196A (en) | 1985-09-06 | 1985-09-06 | Purification and activation of proteins from insoluble inclusion bodies |
| US773,174 | 1985-09-06 | ||
| CA000517604A CA1267496A (en) | 1985-09-06 | 1986-09-05 | Purification and activation of proteins from insoluble inclusion bodies |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA1267496C CA1267496C (en) | 1990-04-03 |
| CA1267496A true CA1267496A (en) | 1990-04-03 |
Family
ID=25097436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000517604A Expired CA1267496A (en) | 1985-09-06 | 1986-09-05 | Purification and activation of proteins from insoluble inclusion bodies |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US4677196A (en) |
| EP (1) | EP0218374B1 (en) |
| JP (1) | JPH082313B2 (en) |
| AT (1) | ATE73138T1 (en) |
| CA (1) | CA1267496A (en) |
| DE (1) | DE3684078D1 (en) |
| DK (1) | DK167192B1 (en) |
| IE (1) | IE60644B1 (en) |
| IL (1) | IL79957A (en) |
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|---|---|---|---|---|
| BG49718A3 (en) * | 1983-07-15 | 1992-01-15 | Bio Technology General Corp | Method for preparing of polypeptid with superoxiddismutasne activitty |
| US5792450A (en) * | 1985-02-05 | 1998-08-11 | Chiron Corporation | Purified human CSF-1 |
| US4766224A (en) * | 1985-08-19 | 1988-08-23 | International Minerals & Chemical Corp. | Purification and activation of proteins from insoluble inclusion bodies |
| US4929700A (en) * | 1987-04-16 | 1990-05-29 | Cetus Corporation | Production of purified, biologically active, bacterially produced recombinant human CSF-1 |
| CA1339757C (en) * | 1987-04-16 | 1998-03-17 | Robert F. Halenbeck | Production of purified biologically active, bacterially produced recombinant human csf-1 |
| CA1305285C (en) * | 1987-04-21 | 1992-07-14 | Malcolm Roy Brandon | Production of proteins in active forms |
| US4931543A (en) * | 1987-05-11 | 1990-06-05 | Cetus Corporation | Process for recovering microbially produced interleukin-2 |
| US5162507A (en) * | 1987-05-11 | 1992-11-10 | Cetus Corporation | Process for recovering purified, oxidized, renatured recombinant interleukin-2 from microorganisms |
| US4801691A (en) * | 1987-05-15 | 1989-01-31 | International Minerals & Chemical Corp. | Method for removing sodium dodecyl sulfate from sodium dodecyl sulfate solubilized protein solutions |
| AU609824B2 (en) * | 1987-06-15 | 1991-05-09 | Southern Cross Biotech Pty Ltd. | Production of proteins in active forms |
| US4981952A (en) * | 1988-10-04 | 1991-01-01 | Eli Lilly And Company | Method for the purification of vitamin K-dependent proteins |
| US5094960A (en) * | 1988-10-07 | 1992-03-10 | New York Blood Center, Inc. | Removal of process chemicals from labile biological mixtures by hydrophobic interaction chromatography |
| EP0365858B1 (en) * | 1988-10-26 | 1994-11-30 | American Cyanamid Company | Process for reducing the aggregate content of growth hormone-like materials |
| GB8825183D0 (en) * | 1988-10-27 | 1988-11-30 | Atomic Energy Authority Uk | Recovery of substances |
| US5064943A (en) * | 1988-12-16 | 1991-11-12 | American Cyanamid Company | Method for solubilization and naturation of somatotropin |
| US5101018A (en) * | 1989-06-12 | 1992-03-31 | International Minerals & Chemical Corp. | Method for recovering recombinant proteins |
| US4975529A (en) * | 1989-08-18 | 1990-12-04 | Monsanto Company | Method of folding somatotropins |
| US5047511A (en) * | 1989-08-28 | 1991-09-10 | Pitman-Moore, Inc. | Method for recovering recombinant proteins |
| US5204256A (en) * | 1989-09-06 | 1993-04-20 | Behringwerke Aktiengesellschaft | Process for the purification of plasminogen activator inhibitor 2 (pai-2) |
| DK580789D0 (en) * | 1989-11-20 | 1989-11-20 | Novo Nordisk As | PROCEDURE FOR PURIFICATION OF POLYPEPTIDES |
| US5605691A (en) * | 1992-09-17 | 1997-02-25 | Ophidian Pharmaceuticals, Inc. | Immunologically active proteins from inclusion bodies |
| US5563057A (en) * | 1994-10-31 | 1996-10-08 | Wisconsin Alumni Research Foundation | Method for refolding misfolded enzymes with detergent and cyclodextrin |
| US5861372A (en) * | 1996-02-22 | 1999-01-19 | The Children's Medical Center Corporation | Aggregate angiostatin and method of use |
| WO1997004003A2 (en) * | 1995-07-21 | 1997-02-06 | Amgen Inc. | Process for protein refolding by means of buffer exchange using a continuous stationary phase capable of separating proteins from salts |
| US6346510B1 (en) | 1995-10-23 | 2002-02-12 | The Children's Medical Center Corporation | Therapeutic antiangiogenic endostatin compositions |
| KR100392780B1 (en) * | 1996-12-04 | 2003-11-01 | 주식회사 엘지생명과학 | Purification method of erk-2 expressed in e. coli |
| ZA9711733B (en) * | 1996-12-31 | 1998-07-01 | Monsanto Co | Method for solubilization and naturation of somatotropins |
| WO1998030684A1 (en) * | 1997-01-08 | 1998-07-16 | Life Technologies, Inc. | Methods for production of proteins |
| US7153943B2 (en) | 1997-07-14 | 2006-12-26 | Bolder Biotechnology, Inc. | Derivatives of growth hormone and related proteins, and methods of use thereof |
| ATE349460T1 (en) * | 1998-07-09 | 2007-01-15 | Barofold Inc | REFOLDING OF PROTEIN AGGREGATES AND INCLUSION PARTICLES BY HIGH PRESSURE |
| ES2290142T3 (en) | 2000-05-16 | 2008-02-16 | Bolder Biotechnology, Inc. | METHODS FOR REPLEGATION OF PROTEINS CONTAINING FREE CISTEINE RESIDUES. |
| EP1434789B1 (en) * | 2000-10-31 | 2008-03-26 | BaroFold, Inc. | Improved protein disaggregation and refolding using high pressure |
| US6936437B2 (en) | 2001-02-23 | 2005-08-30 | Lucia Irene Gonzalez-Villasenor | Methods and compositions for production of recombinant peptides |
| US20070036258A1 (en) * | 2003-09-30 | 2007-02-15 | Osamu Ito | Process for producing radioactive fluorine compound |
| US20090087873A1 (en) | 2006-07-21 | 2009-04-02 | Invitrogen Corporation | Sharply resolving labeled protein molecular weight standards |
| CA2663416A1 (en) * | 2006-09-15 | 2008-03-20 | Barofold, Inc. | High pressure treatment of proteins for reduced immunogenicity |
| EP2102355B1 (en) | 2006-12-14 | 2016-03-02 | Bolder Biotechnology, Inc. | Long acting proteins and peptides and methods of making and using the same |
| SG178870A1 (en) | 2009-08-26 | 2012-04-27 | Commw Scient Ind Res Org | Processes for producing silk dope |
| EP3344651B1 (en) * | 2015-09-02 | 2022-03-02 | Merck Sharp & Dohme Corp. | A process for obtaining insulin with correctly formed disulfide bonds |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4426323A (en) * | 1980-01-10 | 1984-01-17 | Ionics, Incorporated | Selected recovery of proteins from fermentation broths |
| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| US4518526A (en) * | 1982-12-22 | 1985-05-21 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| IE56372B1 (en) * | 1982-12-22 | 1991-07-03 | Genentech Inc | Microbially produced rennet methods for its production and plasmids used for its production |
| US4511503A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
-
1985
- 1985-09-06 US US06/773,174 patent/US4677196A/en not_active Expired - Lifetime
-
1986
- 1986-09-05 EP EP86306871A patent/EP0218374B1/en not_active Expired - Lifetime
- 1986-09-05 DE DE8686306871T patent/DE3684078D1/en not_active Expired - Fee Related
- 1986-09-05 IE IE237086A patent/IE60644B1/en not_active IP Right Cessation
- 1986-09-05 AT AT86306871T patent/ATE73138T1/en not_active IP Right Cessation
- 1986-09-05 IL IL79957A patent/IL79957A/en not_active IP Right Cessation
- 1986-09-05 CA CA000517604A patent/CA1267496A/en not_active Expired
- 1986-09-05 DK DK426386A patent/DK167192B1/en not_active IP Right Cessation
- 1986-09-05 JP JP61208077A patent/JPH082313B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| CA1267496C (en) | 1990-04-03 |
| DK426386D0 (en) | 1986-09-05 |
| JPH082313B2 (en) | 1996-01-17 |
| EP0218374A1 (en) | 1987-04-15 |
| IL79957A (en) | 1991-08-16 |
| IE60644B1 (en) | 1994-08-10 |
| US4677196A (en) | 1987-06-30 |
| DK426386A (en) | 1987-03-07 |
| DK167192B1 (en) | 1993-09-13 |
| EP0218374B1 (en) | 1992-03-04 |
| IL79957A0 (en) | 1986-12-31 |
| DE3684078D1 (en) | 1992-04-09 |
| JPS6269998A (en) | 1987-03-31 |
| ATE73138T1 (en) | 1992-03-15 |
| IE862370L (en) | 1987-03-06 |
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