WO2002018447A1 - Proteine de fusion contenant des acides amines cationiques supplementaires et amelioration d'une bio-operation grace a l'utilisation de ladite proteine - Google Patents

Proteine de fusion contenant des acides amines cationiques supplementaires et amelioration d'une bio-operation grace a l'utilisation de ladite proteine Download PDF

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WO2002018447A1
WO2002018447A1 PCT/KR2001/001394 KR0101394W WO0218447A1 WO 2002018447 A1 WO2002018447 A1 WO 2002018447A1 KR 0101394 W KR0101394 W KR 0101394W WO 0218447 A1 WO0218447 A1 WO 0218447A1
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protein
fusion protein
coli
plyse
enzyme
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PCT/KR2001/001394
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Jin Ho Seo
Dae Hyuk Kweon
Nam Soo Han
Chan Su Rha
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Biongene Co., Ltd.
Shin, Chul, Soo
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Priority to AU2001280223A priority Critical patent/AU2001280223A1/en
Publication of WO2002018447A1 publication Critical patent/WO2002018447A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C12N9/1074—Cyclomaltodextrin glucanotransferase (2.4.1.19)
    • 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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
    • C07K1/1136—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to a cationic fusion gene which can be used to enhance the purification, immobilization and refolding of a desired target protein, a fusion protein encoded therein, an expression vector containing said fusion gene, a microorganism transformed therewith, and processes for purifying, immobilizing and refolding of the target protein.
  • one of the serious problems associated with the methods using recombinant microorganisms is that the expressed protein dose not have the correct tertiary structure and often forms an inactive aggregate which precipitates in the form of an inclusion body. Accordingly, a refolding process is required to recover a protein with biological activity from the inclusion body.
  • refolding processes in general suffer from the problem of low efficiency primarily due to the occurrence of re- precipitation caused by hydrophobic bonding among intermediates, and this problem becomes particularly severe when refolding is conducted at a high protein concentration.
  • the immobilization method which requires least cost is to electrostatically adsorb a charged protein to a carrier having the opposite charge. However, this method has a problem in that the adsorbed enzyme easily desorbs and remains in solution.
  • tags used for the purification and identification of various proteins are such tags as polyhistidine, FLAG peptide, strep-tag, polyaspartic acid, polyarginine, polyphenylalanine, polycystein, calmodulin-binding peptide and green fluorescent peptide, some of which are commercially available.
  • the polyhistidine tag (Novagen, USA), in particular, is widely used in laboratories, however it requires the combined use of an expensive carrier and immobilized metal affinity chromatography(IMAC).
  • Glatz et al. have reported a purification method using a fusion protein prepared by fusing charged anionic amino acid residues to a target protein. They have also been reported: a method using a cationic amino acid, arginine, in the purification step (Brewer, et al., Trends Biotechnol, 3, 119-122(1985)); a method using a cationic amino acid, aspartic acid, in enzyme immobilization(Heng, et al., Biotechnology and Bio engineering, 44, 745-752(1994); Suominen, et al., Biotechnol. Prog, 10, 237-245(1994); and Enzyme Microb.
  • the present inventors have endeavored to develop an improved amino acid fusion system which can be effectively applied to all of the purification, immobilization and refolding processes, and have found that a fusion protein, which comprises more than two consecutive cationic amino acid residues derived from lysine or arginine fused to a desired target protein, can be employed to enhance the efficiencies of the purification, immobilization and refolding processes of the target protein.
  • a fusion gene which can be advantageously employed in the purification, immobilization and refolding processes of a target protein, a fusion protein encoded therein, an expression vector containing said fusion gene, and a microorganism transformed with said expression vector.
  • a fusion protein comprising a target protein and more than two consecutive cationic amino acid residues fused to the target protein, wherein the amino acid is lysine or arginine.
  • a fusion gene encoding said fusion protein and an expression vector containing said fusion gene, pTCGTK6, pRCGTR6, pTCGTKlO, pLPKlO, pLPK12, pTDSBAKlO, pTDSBRlO, pTDSBCKlO, pTDSBCRlO, pTHPPIKl 0 or pTHIPPIRl 0.
  • a microorganism transformed with said vector E. coli BL21(DE3):pLysE:pTCGTK10(KCTC 0842BP), E. coli BL21(DE3):pLysE:pTCGTK6, E. coli BL21(DE3):pLysE:pRCGTR6, E. coli BL21(DE3):pLysE:pLP 10, E. coli BL21(DE3):pLysE:pLPK12, E. coli BL21(DE3):pLysE:pTDSBAK10, E. coli BL21(DE3):pLysE:pTDSBR10, E. coli BL21(DE3):pLysE:pTDSBCK10, E. coli coli BL21(DE3):pLysE:pTDSBCK10, E. coli coli BL21(DE3):pLysE:pTDSBCK10, E. coli BL
  • a process for preparing a fusion protein comprising culturing said microorganism to induce expression of the fusion protein; obtaining a cell extract from the culture solution; and purifying the fusion protein using an ionic matrix.
  • a process for preparing a fusion protein comprising culturing said microorganism to induce expression of the fusion protein in the form of an inclusion body; solubilizing the inclusion body; and obtaining the fusion protein from the solubilized inclusion body using an ionic matrix.
  • a process for preparing an immobilized enzyme comprising binding an ion exchange resin with a fusion enzyme containing a target protein and more than two consecutive cationic amino acid residues fused to the target protein, wherein the amino acid is lysine or arginine.
  • a process for refolding an unfolded protein comprising adsorbing the unfolded fusion protein on an ionic matrix and conducting a refolding reaction.
  • Fig. 1 The procedures for constructing vectors pTCGTK6, pTCGTKlO and pTCGTR6;
  • Fig. 2 Cation exchange resin chromatographic analysis results of recombinant E. coli cell extracts;
  • Fig. 3 Chromatographic and SDS-PAGE analysis results showing the pre-washing effect of recombinant E. coli cell extract
  • Fig. 4 SDS-PAGE analysis result showing the effect of the salt concentration in the cell extract on selective adsorption of CGTKlOase to an exchange resin in the adsorbing step;
  • Fig. 5 Effects of salt concentration on the partition coefficient of CGTKlOase protein:
  • Fig. 6 Effects of salt concentration on the enzyme activity adsorbed on cation exchange column;
  • Fig. 7 Effects of amino acid fusion and immobilization on the pH dependence of the enzyme
  • Fig. 8 Effects of amino acid fusion and immobilization on the stability of the enzyme toward pH changes
  • Fig. 9 Effect of pH on the inactivation rate constants of amino acid- fused enzyme and the immobilized form thereof;
  • Figs. 10A to IOC Thermostabilities of fusion of amino acid-fused enzyme and the immobilized form thereof;
  • Figs. 11 A to 1 ID Continuous production of cyclodextrins using a fixed-bed reactor containing the immobilized enzyme;
  • Fig. 12 The salt concentration-dependent change in the partition coefficient of the enzyme unfolded by urea
  • Fig. 13 The Ca "1" concentration-dependent variations of the refolding efficiencies of free enzyme in solution and immobilized enzyme;
  • Fig. 14 Salt concentration-dependent variations of the refolding efficiencies of enzymes in solution
  • Fig. 15 pH-dependent variations of the refolding efficiencies of enzymes in solution
  • Fig. 16 The salt concentration-dependent change in the refolding efficiency of immobilized enzyme
  • Fig. 17 The pH-dependent change in the refolding efficiency of immobilized enzyme
  • Fig. 18 The protein concentration-dependent variations of . the refolding efficiencies of enzymes in solution;
  • Fig. 19 The protein concentration-dependent change in the refolding efficiency of immobilized enzyme
  • Fig. 20 Chromatograms of the inclusion bodies of CGTKlOase and LPKlOase purified by selectively binding with cation exchange resin;
  • Fig. 21 Chromatograms ofhPPIKlOase and hPPIRlOase purified by using cationic matrix
  • Fig. 22 The protein concentration-dependent change in the refolding efficiency of lipase
  • Fig. 23 The protein concentration-dependent change in the refolding efficiency of disulfide-bond-promoting enzyme
  • Fig. 24 The protein concentration-dependent change in the refolding efficiency of disulfide bond isomerase.
  • Fig. 25 The protein concentration-dependent change in the refolding efficiency of proline isomerase.
  • the inventive fusion protein can be prepared by adding more than 2, preferably 6 to 15, and more preferably 8 to 12 consecutive cationic amino acid residues of lysine or arginine to a desired target protein, usually an enzyme.
  • the cationic amino acid residues can be inserted at the N-terminal or C-terminal of the protein, or anywhere within the protein provided that such insertion does not greatly affect the intrinsic biological function of the protein or enzyme.
  • the term "does not greatly affect the intrinsic biological function of the protein" as used herein refers to a situation that more than 80%, preferably more than 95% of the biological activity of the target protein is maintained after such cationic amino acid fusion.
  • the target protein which may be used in the present invention is inclusive of such enzymes as cyclodextrin glycosyltransferase(CGTase) isolated from Bacillus macerans, lipase from Archaeoglobus fulgidus, disulfide-bond-promoting enzyme, disulfide bond isomerase, proline isomerase and the like, but these do not limit the scope of the invention.
  • CGTase cyclodextrin glycosyltransferase isolated from Bacillus macerans, lipase from Archaeoglobus fulgidus, disulfide-bond-promoting enzyme, disulfide bond isomerase, proline isomerase and the like, but these do not limit the scope of the invention.
  • inventive fusion protein examples include CGTKlOase which has 10 consecutive lysine residues fused to the C-terminal of wild-type(WT) CGTase, and LPK10 and LPK12 having 10 and 12 consecutive lysine residues, respectively, fused to the C-terminal of wild-type lipase.
  • the present invention also provides a fusion gene that encodes the inventive fusion protein.
  • a fusion gene that encodes the inventive fusion protein.
  • the present invention also includes polynucleotides having substantially homologous base sequences with said gene.
  • the fusion protein of the present invention can be obtained by a process which comprises the steps of synthesizing a gene encoding said fusion protein, inserting to a suitable vector to obtain an expression vector, transforming a suitable host, for example, yeast, E. coli. and the like with said expression vector, and culturing the transformed microorganism under an appropriate culture condition.
  • CGTase fusion protein can be prepared by producing an expression vector pTCGTKlO containing a fusion gene which carries 10 lysine residues inserted in the 3 '-end of WT CGTase gene of vector pTCGTl (Lee, K. C. P and B. Y. Tao., Starch, 46, 67-74(1994)), transforming E. coll, for example, E. coli. BL21(DE3) using said expression vector and culturing the same.
  • WT CGTase does not adsorb to a cation exchange resin(pH 7.4) because its theoretical pi value is about 5.0.
  • CGTKlOase which carries fused cationic amino acid residues readily adsorbs to a cationic exchange resin, and thus can be purified thereby.
  • the present invention provides a process for purifying the inventive fusion protein which comprises the steps of culturing a transformed microorganism to induce the expression of the fusion protein, obtaining a cell extract from the culture solution and purifying the fusion protein using a cationic matrix; or culturing the transformed microorganism to induce the expression of the fusion protein and recovering the fusion protein from an inclusion body using a cationic matrix.
  • the cationic matrix used in the present invention may be one of those known in the art, e.g., SP sepharose, S sepharose and CM sepharose, preferably SP sepharose, but these do not limit the scope of the invention.
  • E max as the optimum NaCl concentration at which the maximum activity of a protein is observed during purification by cation exchange chromatograph
  • E max of CGTKlOase is 580 mM
  • E max s of CGTK6ase, and CGTR6ase, which have 6 fused lysine- and arginine residues, respectively are 345 mM and 430 mM, respectively.
  • E max of WT CGTase, which does not adsorb to a cation exchange resin is 0.
  • E max gives a measure of the adsorbing strength of a protein, and a high E max value means that the protein tends to adsorb strongly to a cation exchange resin.
  • a fusion protein having a high E max value remains adsorbed at a high salt concentration, and thus, such a protein can be separated from other intracellular proteins which readily desorb at a relatively low salt concentration.
  • E max of the inventive fusion protein depends on the kind and length of the amino acid residues fused to a target protein. At the same length, E max of the fusion protein becomes higher with arginine residues as compared with lysine residues, and if one kind of amino acid is used, E max of the fusion protein becomes higher as the multiplicity increases. Moreover, a fusion protein having a high E max dissociates in solution more sluggishly.
  • the intracellular proteins readily dissociate at a low salt concentration but some other proteins remain adsorbed to a cation exchange resin even at a high concentration of salt, e.g., 100-300 mM, and act as impurities in the final purifying solution.
  • the partition coefficient becomes larger.
  • the partition coefficients of CGTK6ase, CGTR6ase and CGTKlOase, for example, are 2.38, 4.97 and 9.88, respectively.
  • the purity of the desired protein can be raised by pre- washing the column loaded with the adsorbed fusion protein with a buffer solution containing 10 to 1000 mM salt before initiating cation exchange chromatography.
  • This procedure is intended to desorb weakly adsorbed intracellular protein impurities from the column.
  • a purity of more than 95% can be achieved by pre-washing adsorbed crude CGTKlOase with a 400 mM salt solution, according to the results of SDS-PAGE and densitometer analyses, or about 98% when the specific enzyme activity is compared with a pure protein standard obtained by affinity chromatography.
  • This method can thus be used to obtain the desired protein having an improved purity.
  • the present invention provides a simple means to produce an enzyme which can be directly used without further purification.
  • the purification efficiency of the fusion protein can be enhanced by controlling not only the dissociation step as described above but also the adsorption step.
  • This mode of procedure may be represented by a term "selective binding", which provides an additional means for enhancing the recovery and purity of the desired protein.
  • Intracellular proteins compete with the desired protein in the process of binding to ion exchange resin, and thus, when the adsorption step is carried out in the presence of a salt at a concentration in the range of 100-500 mM, the binding of some of the intracellular proteins can be inhibited, thereby enhancing the adsorption efficiency and purity of the desired protein.
  • the fusion protein of the present invention can be used in the preparation of an immobilized enzyme by way of electrostatic adsorption to a cation exchange resin.
  • an immobilized enzyme for example, in case of CGTase, more than 90% of the original enzyme activity is maintained after CGTase is fused with cationic amino acid residues and the fused protein retains its full activity even when it is immobilized.
  • the immobilization method of the present invention is much simpler than those described in the prior arts.
  • the inventive immobilized enzyme which is fully active and does not leach out, can be used in an immobilized enzyme fixed-bed reactor.
  • the inventive fusion protein and an immobilized form thereof have similar pH dependencies as WT enzyme. Further, although the fusion enzyme in solution has comparable stability toward pH changes as WT enzyme, the immobilized enzyme has much higher pH stability.
  • the inventive fusion enzyme is lower than WT enzyme but the immobilized fusion enzyme is much higher.
  • a desired product can be efficiently produced from a suitable substrate by operating an enzyme reactor containing the inventive immobilized enzyme.
  • inventive fusion protein also provides a means to enhance the refolding and other process step efficiencies. This can be accomplished by removing the protein unfolded by the action of a denaturing agent, immobilizing on a cation exchange resin, and inducing refolding. Further, the present invention provides an efficient means to refold a protein at a high protein concentration without the risk of protein aggregation.
  • Example 1 Construction of a expression vector containing the fusion protein having cationic amino acid
  • the PCR program consisted of 1 minutes of denaturation using ImM MgCl 2 at 95 °C, 2 minutes of annealing at 48 °C, 3 minutes of polymerization at 72 °C for 30 cycles, and 5 minutes of final polymerization at 72 °C.
  • the initial denaturation reaction was carried out at 95 °C for 5 minutes.
  • Fig. 1 shows the procedure for constructing expression vectors pTCGTK6, pTCGTR6 and pTCGTKlO.
  • PCR was carried out using vector pELP-1 as a template and 5'- primer of SEQ ID NO : 11 and 3 '-primer of SEQ ID NO : 12 or 13 in accordance with the same method described in (1), wherein pELP-1 is a vector derived by inserting the lipase gene of Archaeoglobus fulgidus(ATCC 49558D) at the Ndel/BamUl section of pET3a vector(Novagen, U.S.A).
  • the DNA fragment thus obtained was treated with NdeI(NEB, England) and _5 ⁇ HI(NEB, England), and ligated with the fragment obtained by cleaving Vector pET3a(Novagen, USA) with the same restriction enzymes, to construct plasmids pLPKlO and pLPK12 which contained nucleotides encoding 10 and 12 consecutive lysines, respectively.
  • 5 '-primer of SEQ ID NO : 14 and 3 '-primer of SEQ ID NO : 15 were mixed and annealed at room temperature, and then, treated with polynucleotide kinase(NEB, USA) at 37°C for 1 hour.
  • pET29-b(Novagen, USA) was cleaved with Xhol and BamHI and ligated with said annealed DNA fragment to construct plasmid pETKlO.
  • pETRlO was prepared by using 5 '-primer of SEQ ID NO : 16 and 3 '-primer of SEQ ID NO : 17.
  • PCR was carried out using human proline isomerase cDNA(ATCC 78809) as a template and primers of SEQ ID NOS : 18 and 19 and the product DNA fragment was treated with Ndel NEB, England) and _5a HI(NEB, England). Further, in order to obtain disulfide- bond-promoting enzyme, PCR was carried out using the chromosome of E. coli. as a template and primers of SEQ ID NOS : 20 and 21 and the product DNA fragment was treated with the same restriction enzymes. Moreover, in order to obtain disulfide bond isomerase, PCR was carried out using the chromosome of __?. coli as a template and primers of SEQ ID NOS : 22 and 23 and the product DNA fragment was treated with the same restriction enzymes.
  • vectors pETKlO and pETRlO obtained above were each treated with the same restriction enzymes, and ligated, to construct plasmids pDSBAKlO, pDSBCKlO, pHPPIKlO, pDSBARlO, pDSBCRlO and pHPPIRlO, each containing nucleotides encoding 10 consecutive lysines or arginines.
  • E. coli BL21(DE3):pLysE(Novagen, U.S.A.) was transformed with expression vector pTCGTKlO prepared in Example 1 and transformed colonies were selected in a plate containing ampicillin, to obtain a transformant, E. coli BL21(DE3):pLysE:pTCGTK10, which was deposited with Korean Collection for Type Cultures (Korea Research Institute of Bioscience and Biotechnology) on July 21, 2000 under accession number KCTC 0842BP.
  • E. coli BL21(DE3):pLysE:pTCGTl which produces WT CGTase(a control)
  • E. coli BL21(DE3):pLysE:pTCGTK6 transformed with expression vector pTCGK6
  • E. coli BL21(DE3):pLysE:pTCGTR6 transformed with expression vector pTCGTR6 were each incubated in 5 no. of LB medium(containing 50 mg/i of ampicillin) overnight at 37 °C, and then, 1 no. of the culture solution was re-incubated in 100 ⁇ of LB medium containing 2 g/i of glucose at 30 ° C .
  • Fig. 2 shows the results of such analyses conducted during cation exchange resin chromatography of recombinant E. coli cell extracts, wherein A, B, C and D are results for WT CGTase, CGTK6ase, CGTR6ase and CGTKlOase, respectively, and the lines, ( ), ( - ), and ( - - ) represent UV absorption at 280nm, gradient and conductivity, respectively, while the bar denotes enzyme activity. As shown in Fig.
  • Figs. 3b, 3c and 3d represented similar properties except for the enzyme activity.
  • pKa of lysine is 10.0 and that of arginine, 12.0. It was confirmed that the adsorbing strength of the fusion protein having arginine residues was higher than that having lysine residues when the lengths of the lysine and arginine tags are the same.
  • the binding strength of CGTKlOase was higher than those of CGTR6ase and CGTK6ase. The result is shown in table 1.
  • Example 3 Improvement of purity of a fusion protein through Pre- washing
  • CGTK6ase, CGTR6ase and CGTKlOase were obtained as in Example 2 and subjected to cation exchange chromatography as follows.
  • the cell extract was loaded into the same column and pre-washed with a phosphate buffer solution containing 0, 150, 200 or 400 mM NaCl, and then, an eluate .was introduced thereto.
  • the resulting solution was subjected to 12% SDS-PAGE using the eluate and a gel was dyed with coomassie blue.
  • Fig 3 shows effects of pre-washing recombinant E. coli cell extracts on the chromatogram and SDS-PAGE analysis, wherein A represents CGTKase pre-washed with 150 mM NaCl; B, WT CGTase pre-washed 200 mM NaCl; C, CGTR6ase pre-washed with 200 mM NaCl; D, CGTKlOase pre-washed with 400 mM NaCl; and S, standard molecular protein. WT CGTase did not adsorb even when the buffer contained no salt.
  • CGTKlOase remained adsorbed in even 400 mM NaCl and the intracellular proteins except the fusion protein were removed(Fig. 3D). Some intracellular proteins which were strongly adsorbed to the resin can not be removed through pre-washing with 300 mM NaCl but can be mostly removed at 400 mM. These results are in line with the optical density scans at 280 nm: the higher the salt concentration used in pre-washing, the lower the protein concentration recovered by desorbing with 1 M salt. This is due to the removal of intracellular proteins through pre-washing.
  • Example 4 Improvements of purity and efficiency through selective adsorption
  • S is a standard molecular protein
  • A the protein purified by affinity chromatography (Sundberg, L. and Porath J., J. of Chromatogr., 90, 87-98(1974)
  • B the protein purified by pre-washing with 400 mM salt followed by cation exchange chromatography in accordance with the method described in Example 3
  • C D, E, F and G, proteins obtained by adsorbing the cell supernatants containing 0, 100, 200, 300 and 400 mM salt, respectively.
  • the recovery and purification efficiencies can be increased, as observed in D, E, F, and G, by inhibiting the adsorption of intracellular proteins by way of raising the salt concentration at the adsorption step from 0 to 300 mM.
  • the adsorption of intracellular proteins which usually adsorb more weakly than the fusion protein is selectively inhibited by added salt at the adsorption step. .
  • step 1 Partition coefficient( ⁇ )s were measured at various salt concentrations as follows. A purified CGTKlOase solution was equilibrated(20 mM phosphate buffer solution, pH 7.0) to an ionic concentration of 0 to 500 mM and mixed with a resin(SP-Sepharose, Pharmacia, Sweden) equilibrated at the same concentration to immobilize the fusion enzyme CGTKlOase. The relative amounts of the protein adsorbed and the protein remaining in solution were measured to determine the partition coefficient, according to Equation 1, wherein q is the concentration of the protein adsorbed and p is the concentration of the protein in solution.
  • Fig. 5 illustrates the salt concentration-dependant change in the partition coefficient of CGTKlOase protein.
  • the partition coefficient is about 0.95 at a salt concentration range of 0 to 300 mM.
  • the fusion enzyme CGTKlOase was immobilized by ionic interaction rather than by covalent bonding.
  • WT CGTase, non-immobilized CGTKlOase and an immobilized form thereby were each added to a phosphate buffer solution having a pH in the range of 4 to 10, kept at 50 °C for 1 hour and the enzyme activity was measured. The result is expressed by a value relative to a maximum value of 100.
  • WT CGTase and CGTKlOase all exhibit similar trends and the immobilized enzyme was found to be stable at a wide pH range.
  • the enzyme inactivation process can be represented by Equation 2, wherein A is remaining enzyme activity; t, time; and k, inactivation rate constant.
  • Example 8 Measurment of thermostability of immobilized enzyme
  • thermostability of enzymes in 20 mM phosphate buffer (pH 7.0) was examined at 50 ° C and 25 ° C , and the result is shown in Fig. 10.
  • Figs. 10a and 10b show the time-dependent activities of the fusion protein and immobilized fusion protein at 50 °C and 25 °C, respectively, which shows that the thermostability of CGTKlOase was lower than free WT CGTase in solution while the immobilized CGTKlOase exhibited enhanced stability that was equal or higher than that of WT CGTase.
  • thermostability was measured at 50 °C in presence of 5 mM CaCl 2 in 20 mM MOPS(3-[N- Morpholinojpropanesulfonic acid, Sigma, U.S.A.)(pH 7.0). The result in Fig 10c shows that the thermostability of the immobilized enzyme was markedly enhanced.
  • Example 9 Stability of immobilized enzyme in a bio-process
  • the reactor column was kept at 25 °C using a water jacket, and a reactant solution, which was prepared by adding lOg/ ⁇ of soluble starch to the same phosphate buffer containing 5mM CaCl 2 , was continuously introduced to the column using a gear pump at a rate of 0.5 no ⁇ /min.
  • Cyclodextrin is composed of ⁇ -CD, ⁇ - CD and V -CD which are composed of 6, 7 and 8 glucose units, respectively.
  • a TLC plate(KF5, Whatman, USA) was activated in a 110 °C oven for 1 -2 hours before use. Each sample was diluted to the concentration range of control standards, and then, 1- 2 ⁇ & of the resulting solution and standards were applied to the plate.
  • the standards contained 0.005 - 0.05% glucose, maltose, maltotriose and known amounts of the three forms of CD.
  • the plate was dried and developed twice in a chamber containing a solvent mixture (nitromethane:water:n-propanol, 2:1.5:4, v/v). Then, the plate was kept in a 110 °C oven for 10 minutes until the smell of the solvent was not detectable.
  • Fig. 11 shows the amount of CD produced by the immobilized enzyme, wherein A, B, C and D represent -CD, ⁇ -CD and y -CD and total CD, respectively. As shown in Fig. 11, a steady amount of CD can be produced using the immobilized enzyme over a long period of time without any significant changes in the relative amounts of tliree forms of CD produced.
  • an immobilized enzyme can be efficiently prepared by fusing cationic amino acid residues to a target enzyme, and a desired product can be produced using said immobilized enzyme.
  • the precipitated resin was washed with the same solution, and then, a 17 x volume amount of 20 mM phosphate buffer solution(pH 7.0) containing 1M NaCl and 9M urea was added thereto to release the protein adsorbed on the resin.
  • the concentration of the eluted protein in solution was measured and the concentration of the protein adsorbed on the resin was calculated (designated q).
  • the partition coefficient was then assessed in accordance with Equation 2 described in Example 5.
  • the result shown in Fig. 12 depicts that the partition coefficient was more than 0.9 in 1- 100 mM NaCl but it dropped precipitously at higher salt concentrations to nearly 0 at 400mM. Thus, it was confirmed that a denatured fusion enzyme could also be easily immobilized.
  • CGTKlOase was allowed to stand at room temperature in 20 mM MOPS buffer (pH 7.0) containing 9M urea for 4 hours to be denatured, mixed with SP-sepharose pre-equilibrated under the same condition to be immobilized, and then, unreacted, free enzyme was removed by washing.
  • Free CGTKlOase in solution and the immobilized enzyme were added to refolding buffer (20 mM MOPS buffer solution, pH 7.0) to concentrations of 50 mg/no. and 1 mg/m ⁇ , respectively. After dilution(the concentration of urea was 0.45 M in both cases), and the diluted solution containing free enzyme was allowed to stand at 15 ° C for 16 hours and the enzyme activity was measured.
  • the protein concentration was measured after desorbing with 1M NaCl.
  • Specific enzyme activity refers to the value obtained by dividing enzyme activity by the protein concentration, and the refolding efficiency was calculated based on the specific enzyme activity (100%) of WT enzyme.
  • Refolding reactions were carried out at various NaCl concentrations in the range of 0 to 400mM using free WT CGTase in solution, free CGTKlOase or a mixture of SP-sepharose resin and WT CGTase, in accordance with the method described in (1).
  • WT CGTase which does not bind to a resin was used as a control for examining the influence of the resin on the refolding efficiency caused by resin's interaction with CGTase' s protein segments other than lysine.
  • WT CGTase as a whole was not influenced by NaCl concentration and the efficiency decreased slightly when the resin was present.
  • the refolding efficiency of CGTKlOase was higher than that of WT CGTase and greatly influenced by NaCl concentration.
  • the refolding efficiency increased when WT enzyme or the fusion enzyme was refolded in solution.
  • Refolding of the immobilized CGTase was examined using refolding solutions containing various amounts of NaCl in the range of 10-400mM, in accordance with the method described in (1).
  • Fig. 16 displays the NaCl concentration-dependant variation of the refolding efficiency of the immobilized CGTKlOase. As shown in Fig. 16, the refolding efficiency calculated based on the specific enzyme activity of the refolded enzyme was 100% over the entire NaCl concentration range examined. However, as the solid circles of Fig. 16 show, the recovery rate of the protein was lower at a higher salt concentration, possibly due to the presence of some ionized protein which remains in solution.
  • Fig. 17 displays the pH-dependent change in the refolding efficiency of the immobilized CGTKlOase. As shown in Fig. 17, the refolding efficiency of the immobilized CGTKlOase was in most cases much higher than WT CGTase observed in (2) or free CGTKlOase in solution. The amount of remaining protein was almost constant at pH 6.0 - 8.5 but the refolding efficiency was greatly influence by pH.
  • Refolding reactions were carried out using free WT CGTase, free CGTKlOase in solution and the immobilized CGTIOase at various protein concentrations in the range of 0.004 - 8 mg/irv. , in accordance with the method described in (1).
  • Figs. 18 and 19 display the protein concentration-dependent variations of the refolding efficiency of free enzyme in solution and immobilized enzyme, respectively.
  • Fig. 18a shows the activity of recovered enzyme
  • Fig. 18b the recovered specific enzyme activity.
  • the refolding efficiency of the free enzyme in solution decreased with increasing protein concentration. This result is due to the occurrence of increasing re-aggregation between refolded intermediates or denatured proteins at a high concentration.
  • a protein concentration of more than 0.1 mg/m 5 WT CGTase exhibited a refolding efficiency of 4%, and CGTKlOase, an efficiency of only 7%.
  • Fig. 18a shows the protein concentration-dependent variations of the refolding efficiency of free enzyme in solution and immobilized enzyme, respectively.
  • Fig. 18a shows the activity of recovered enzyme
  • Fig. 18b the recovered specific enzyme activity.
  • the refolding efficiency of the free enzyme in solution decreased with increasing protein concentration. This result is due to the
  • the fusion protein immobilized through the fused lysine moiety showed a refolding efficiency of 100% at a high protein concentration of even 8 mg/m ⁇ .
  • the refolding of the fusion protein immobilized through the fused lysine moiety occurs with a 25-fold higher refolding efficiency even at a 80-fold higher protein concentration. This represents a 2,000-fold enhancement of the process productivity.
  • the free enzyme in solution gives only a low refolding efficiency at a low protein concentration while the immobilized enzyme undergoes refolding with a high efficiency even at a high protein concentration. This is due to fact that the free enzyme in solution tends to undergo by re-aggregation between refolding intermediates, while such re- aggregation is inhibited in the case of the immobilized enzyme.
  • Example 12 Purification of lipase containing lysine and CGTKlOase expressed as inclusion body
  • E. coli BL21(DE3):pLysE(Novagen, U.S.A.) was transformed with each of the expression vectors pLPKlO and pLPK12 prepared in Example 1 (2) and the transformed colonies were selected from a plate containing ampicillin.
  • E. coli BL21 (DE3):pLysE:pTCGTKl 0 which produces GCTK1 Oase,
  • E. coli BL21(DE3):pLysE:pELP-l which produces WT lipase E. coli BL21(DE3):pLysE:pLPK10 and E. coli BL21(DE3):pLysE:pLPK12 were each incubated in 5 n ⁇ i of LB medium(containing 50 >l& of ampicillin) overnight at 37 °C , and then, 1 og of the culture solution was re-incubated in lOOm ⁇ of LB medium containing 2 g/i of glucose at 37°C . After incubating for 4 hours until glucose was exhausted, 1 mM IPTG was added thereto to induce protein expression.
  • the resultant solution was centrifuged to obtain cell mass, which was suspended in 50mM phosphate buffer solution (pH 7.4) and disrupted using a French pressure (Aminco, U.S.A.).
  • the supernatant containing soluble cell extracts was removed by centrifugation and the isolated precipitate was dissolved in 9M urea(20 mM MOPS buffer solution, pH 7.0).
  • the solubilized protein was purified by the selective binding method described in Example 4 and subjected to SDS- PAGE. The result is shown in Fig. 20. In Fig.
  • S represents a standard molecular weight protein
  • lanes 1 and 5 are inclusion body samples recovered from E: coli BL21(DE3):pLysE:pTCGTK10 and E. coli BL21(DE3):pLysE:pLPK10, respectively.
  • Lanes 2, 3, 4, and Lanes 6, 7, 8 correspond, respectively, to enzyme solutions purified by selective binding the inclusion bodies recovered from E. coli BL21(DE3):pLysE: ⁇ TCGTK10 and E. coli BL21(DE3):pLysE:pLPK10 in 300, 350 and 400 mM NaCl, washing with the same salt solutions, and desorbing with 1M NaCl.
  • Example 13 Purification of disulfide-bond-promoting enzyme, disulfide bond isomerase and proline isomerase, each containing 10 lysines and arginines
  • E. coli BL21(DE3):pLysE(Novagen, U.S.A.) was transformed with each of the expression vectors pDSBAKlO, pDSBCKlO, pHPPIKlO, pDSBSRlO, pDSBCRlO and pHPPIRlO prepared in Example 1 (3) and transformed colonies were each selected from a plate containing ampicillin to obtain E. coli BL21(DE3):pLysE:pDSBAK10, E. coli BL21(DE3):pLysE:pDSBCK10, E. coli BL21(DE3):pLysE:pHPPIK10, E.
  • Disulfide-bond-promoting enzyme disulfide bond isomerase and proline isomerase, each containing 10 lysines and arginines, were purified in accordance with the method described in Example 3. As the result in Fig. 21 shows, these enzymes can be effectively purified by exploiting the fused cationic amino acid residues, as in the case of CGTase.
  • Example 14 Refolding of disulfide-bond-promoting enzyme, disulfide bond isomerase and proline isomerase immobilized on a solid through cationic amino acid fusion system
  • Disulfide-bond-promoting enzyme disulfide bond isomerase, each containing fused cationic amino acid residues, were immobilized on a resin and subjected to refolding as in Example 11 and the results are depicted in Figs. 22, 23, 24 and 25.
  • the protein refolding proceeded smoothly without the occurrence of protein aggregation even at a protein concentration of more than 1 mg/no ⁇ . This process is advantageous over refolding in solution in that the process volume can be reduced while enhancing the refolding efficiency.

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Abstract

L'invention concerne une protéine de fusion contenant plus de deux restes d'acides aminés cationiques consécutifs sélectionnés dans le groupe comprenant la lysine et l'arginine et ajoutée à une protéine désirée, permettant d'améliorer l'efficacité de purification, d'immobilisation et de renaturation de la protéine désirée.
PCT/KR2001/001394 2000-08-18 2001-08-17 Proteine de fusion contenant des acides amines cationiques supplementaires et amelioration d'une bio-operation grace a l'utilisation de ladite proteine WO2002018447A1 (fr)

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KR1020000047768A KR20020014459A (ko) 2000-08-18 2000-08-18 양이온성 아미노산이 부가된 융합단백질 및 이를 이용한생물공정의 개선방법

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Cited By (2)

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US7858338B2 (en) 2006-10-13 2010-12-28 Novo Nordisk Health Care Ag Processing enzymes fused to basic protein tags
US8137929B2 (en) 2005-04-15 2012-03-20 Novo Nordisk Health Care Ag Basic protein purification tags from thermophilic bacteria

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KR100494644B1 (ko) * 2002-07-25 2005-06-13 (주)바이오버드 단백질의 산업적인 리폴딩방법
KR100735738B1 (ko) * 2003-12-05 2007-07-06 학교법인 인하학원 Ｓｍｂ 크로마토그래피를 이용하여 단백질을 재접힘시키는방법
KR102042741B1 (ko) 2017-09-14 2019-11-08 (주)바이오액츠 영상화용 링커 화합물

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WO1998059241A1 (fr) * 1997-06-20 1998-12-30 Bio Merieux Procede de mise en evidence d'un materiel biologique cible, phase de capture, phase de detection et reactif les contenant

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8137929B2 (en) 2005-04-15 2012-03-20 Novo Nordisk Health Care Ag Basic protein purification tags from thermophilic bacteria
US8426566B2 (en) 2005-04-15 2013-04-23 Novo Nordisk Healthcare Ag Basic protein purification tags from thermophilic bacteria
US7858338B2 (en) 2006-10-13 2010-12-28 Novo Nordisk Health Care Ag Processing enzymes fused to basic protein tags
US8206959B2 (en) 2006-10-13 2012-06-26 Novo Nordisk Health Care Ag Processing enzymes fused to basic protein tags
US8283146B2 (en) 2006-10-13 2012-10-09 Novo Nordisk Health Care Ag Processing enzymes fused to basic protein tags

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