WO2009005201A1 - Procédé de fabrication du facteur 1a issu de cellules stromales comme forme de protéine soluble active - Google Patents

Procédé de fabrication du facteur 1a issu de cellules stromales comme forme de protéine soluble active Download PDF

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WO2009005201A1
WO2009005201A1 PCT/KR2008/000265 KR2008000265W WO2009005201A1 WO 2009005201 A1 WO2009005201 A1 WO 2009005201A1 KR 2008000265 W KR2008000265 W KR 2008000265W WO 2009005201 A1 WO2009005201 A1 WO 2009005201A1
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protein
sdf
expression vector
coli strain
column
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Chang Ill Ban
Tae Gyun Kim
Do Wook Ryu
Kyo Han Ahn
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POSTECH Academy Industry Foundation
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    • 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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • 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
    • 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
    • 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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof

Definitions

  • the present invention relates to a method for the production of an active form of human stromal-derived factor (SDF-I a). More particularly, the present invention relates to a method for renaturing an inclusion body of proteins expressed in a large quantity in E. coli into an active form using a column.
  • SDF-I a human stromal-derived factor
  • Proteins are large organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. After being translated, a protein undergoes a change in three-dimensional conformation to a biologically active form or a thermodynamically stable form through a folding process in which relatively weak forces such as hydrogen bonds, ionic bonds, etc. are involved.
  • proteins are isolated or extracted in thermodynamically stable or biologically active forms from various animals and plants.
  • inclusion body refers to an insoluble aggregate of proteins in which the native three-dimensional structure thereof is not formed due to protein misfolding. Typically, an inclusion body is formed as a result of misfolding due to non- covalent interactions between hydrophobic residues exposed from dynamic intermediates in folding process (Cleland, Tl. L., Wang, I. C, Biochemistry, 29, 1 1072-1 1078, 1990).
  • An inclusion body which is a protein aggregate with a modified three- dimensional structure, can be converted into a native form, which has biological activity.
  • the protein aggregate is solubilized in a buffer containing a denaturant to unfold the polypeptide chain and the denaturing condition is removed through dilution or dialysis to refold the protein into a native form.
  • a refolding process by which a recombinant protein existing as an inclusion body can be recovered in an active form generally comprises solubilizing an inclusion body with a denaturant and removing the denaturant to induce correct folding of the protein.
  • the removal of denaturants may be implemented by simple dilution, serial dilution, dialysis, gel filtration chromatography, etc.
  • Simple dilution, a typical process for protein refolding, suffers from the following disadvantages.
  • a large reactor is required for reaction because a sample must be diluted to 4 to 100 volumes. With a limitation only to a very low concentration of proteins, a simple dilution method shows low efficiency. Further, a concentration process is required due to the large volume to be treated, increasing a cost for the production and purification of the recombinant protein.
  • disulfide bonds may be formed randomly during simple dilution.
  • a protein refolding method using size exclusion batch chromatography was developed and utilized. Based on column chromatography, this protein refolding method is advantageous in that a buffer can be easily exchanged with a desired one and the protein denaturant used in protein refolding can be readily removed.
  • size exclusion batch chromatography allows a refolding process to be applied to a protein concentration of 80 mg/ml or more, whereas simple dilution is impossible to apply to a concentration of 10 mg/ml.
  • a size exclusion batch chromatography method can be automated for the full process thereof, thereby affording process standardization and effective management.
  • the refolding process employs a urea-containing buffer.
  • Urea plays an important role in refolding proteins.
  • the recombinant proteins are aggregated to form inactive inclusion bodies in many cases.
  • urea is widely used as a denaturant.
  • 8 M urea is used for unfolding inclusion bodies produced in recombinant E. coli and 2 M urea is used for refolding the denatured polypeptides at a high yield.
  • SDF-I a stromal cell-derived factor 1 a
  • CXC chemokine a small cytokine belonging to the chemokine family, known as CXC chemokine, which was first isolated from a mouse bone marrow stromal cell line (Nagasawa T. et al., Nature 1996, 382, 635-638).
  • SDF-I a is involved in diverse functions including hematopoiesis, cardiogenesis, B cell lymphopoiesis, and cell migration (Doitsidou M. et al., Cell, 2002, 1 1 1 , 647- 659).
  • SDF-I a is known to play an important role in the mechanism of embryo development and division by specifically binding to the membrane receptor CXCR4 (Ma Q., Proc. Natl. Acad. Sci. USA, 1998, 95, 9448-9453).
  • SDF-I a is expressed as an insoluble form in bacterial expression systems. Many research groups have made extensive effort to obtain SDF-I a in an active form.
  • the present invention pertains to a method for the renaturation of an inclusion body of arecombinant protein into an activated, soluble form.
  • a recombinant SDF-I a protein when expressed in the form of an inclusion body, it is renatured into an active form through a protein refolding system using a column.
  • SDF-I a is cloned into an expression vector containing a 6x His-tag and a tobacco etch virus (TEV) protease recognition site so that a recombinant SDF-I a is expressed, with 6x His residues tagged at the N terminus thereof and a TEV recognition site subsequent to the tag.
  • TEV tobacco etch virus
  • TEV protease recognition site As for the TEV protease recognition site, it is digested with the enzyme so as to remove the 6x His residues attached thereto, leaving activated, soluble SDF-I a alone.
  • the present invention pertains to an activated, soluble SDF-I a protein produced by the method.
  • the SDF-I a produced by the method was found to be activated, as assayed for the characteristic cell chemotaxis thereof.
  • a recombinant SDF-I a protein is expressed.
  • a gene coding for SDF-I a may be obtained by PCR amplification from a human fibroblast cDNA library.
  • the SDF-I a gene is cloned into an expression vector containing a 6x His tag and a tobacco etch virus (TEV) protease recognition site so that a modified SDF-I a protein containing 6x His residues at an N-terminal region thereof followed by a tobacco etch virus protease recognition site is expressed.
  • TSV tobacco etch virus
  • Useful as the expression vector in the present invention is a pET-28a vector.
  • both the genetic materials are digested with BamHI and Xhol, and then react with each other in the presence of a ligase to afford a recombinant expression vector.
  • the recombinant expression vector thus obtained is transformed into an ER2566 competent cell (Novagen) to produce a transformant which anchors the expression vector therein.
  • the recombinant pET-28a vector carrying an SDF-I a gene is introduced into E. coli BL21 (DE3).
  • E. coli BL21 E. coli BL21 (DE3).
  • a mutant SDF-I a (K1 A) gene is synthesized by polymerase chain reaction using site-directed mutagenesis.
  • the mutant gene is transformed into E. coli BL21 (DE3) to express the mutant protein.
  • an affinity column such as a HiTrap Chelating HP column (GE Healthcare)
  • a HiTrap Chelating HP column GE Healthcare
  • the SDF-I a protein is immobilized on a HiTrap Chelating HP column resin via the binding of the 6x His tag to the Ni ions of the resin. While being sustained by the column, the SDF-I a is depleted of the urea at a very low rate (0.2 ml/min), with protein refolding taking place gradually.
  • the column onto which the protein is immobilized is sufficiently washed with a 45 mM imidazole buffer, followed by 300 mM imidazole to completely elute the refolded SDF-I a from the column. Subsequently, the 6x His-tag is deleted from the protein by cleavage with
  • a protein may be produced in vivo using a recombinant expression vector carrying a gene encoding the protein.
  • the gene is a nucleotide sequence coding for SDF-I a (stromal cell-derived factor- 1a) and the expression vector is preferably based on pET-28a containing a 6x His tag and a tobacco etch virus (TEV) protease recognition site.
  • the present invention pertains to an E. coli strain anchoring the expression vector therein.
  • the E. coli is based on BL21 (DE3).
  • the present invention pertains to a method for producing SDF- 1 a in an active form in a large quantity, comprising: (a) constructing a recombinant expression vector carrying a nucleotide sequence coding for SDF-I a, a 6x His tag and a tobacco etch virus protease (TEV) protease recognition site; (b) transforming the recombinant expression vector into E. coli and culturing the E.
  • TSV tobacco etch virus protease
  • the unfolding is carried out by lysing the E. coli of step (b) in the presence of a protease inhibitor and treating the resulting cell lysate with a denaturant to give a denatured SDF-I a solution.
  • the denaturant is urea.
  • the recombinant expression vector is based on pET-28a.
  • the E. coli useful in the present invention is BL21 (DE3).
  • the refolding is carried out by loading the denatured SDF-I a solution on an affinity chromatography column pre-equilibrated with Ni and a denaturing buffer, renaturing the SDF-I a protein immobilized on the column with a renaturing buffer substituting for the denaturing solution, washing the renatured SDF-I a protein with an imidazole solution, and eluting an active SDF-I a protein having a 6x His tag with an imidazole buffer.
  • the present invention is concerned with the active SDF-I a protein having a 6x His tag, produced by the method of the present invention.
  • the method further comprises removing the 6x His tag from the active SDF-I a by use of TEV protease after the eluting step.
  • the method further comprises filtering out SDF-I a having a 6x His tag through affinity chromatography after the removing step.
  • the method further comprises purifying SDF-I a free of a 6x His tag using peptide column chromatography after the filtering.
  • the present invention pertains to an active form of the SDF-I a which is free of a 6x His tag.
  • the present invention provides a method for renaturing an inactive form of protein into an active form, comprising: expressing a protein containing a group through which the protein can bind to a column: unfolding the protein and immobilizing the protein onto a column; refolding the immobilized protein; and separating the refolded protein from the column.
  • the protein may be one that is changed in activity through protein unfolding and refolding processes.
  • the method according to the present invention may be applicable to a variety of insoluble proteins which show no activity without renaturation.
  • the protein is SDF-I a, which is produced in an insoluble form upon expression in an E. coli system using an expression vector carrying an SDF-I a gene, and can be refolded into an active, solubilized form through renaturation.
  • the protein preferably contains an attachment group which allows the protein to bind to a column for use in protein unfolding and refolding.
  • the attachment group may be added to a protein of interest by inserting a nucleotide sequence coding for the attachment group, along with a nucleotide sequence coding for the protein, into an expression vector.
  • the attachment group may be introduced into a protein of interest through a chemical reaction therebetween.
  • the method may further comprise removing the attachment group from the protein.
  • the removal of the attachment group may be achieved by introducing a nucleotide sequence coding for an enzyme cleavage site between a protein of interest and the attachment group, expressing the recombinant protein, and cleaving the cleavage site with a suitable enzyme.
  • the expression vector comprises a nucleotide sequence encoding a tobacco etch virus (TEV) recognition site so that a 6x His tag, serving as the attachment group, can be removed after protein refolding.
  • TSV tobacco etch virus
  • the present invention provides a method for producing SDF-I a in an active form, comprising: constructing a recombinant expression vector containing nucleotide sequences respectively coding for SDF-I a, 6x His-tag, and tobacco etch virus (TEV) protease; expressing a recombinant SDF-I a protein in an E. coli strain transformed with the expression vector; unfolding an aggregate of expressed proteins under a denaturing condition; and refolding the recombinant protein on a column.
  • the expression vector is pET-28a and the E. coli strain is based on BL2KDE3), in which the protein is over-expressed in an insoluble form.
  • the bacteria is lysed in the presence of a protease inhibitor, and the cell lysate thus obtained is treated with a denaturant, preferably with urea, so as to unfold protein aggregates.
  • the cell lysate is loaded on a HiTrap Chelating HP column to immobilize the unfolded protein onto the column, followed by refolding the protein thereon. Then, the histidine residues tagged to the N-terminal of the refolded SDF-
  • an SDF-I a gene amplified from a human fibroblast cDNA library is introduced into a recombinant E. coli system for the overexpression thereof, and an inclusion body of the SDF-I a protein thus expressed can be renatured into a soluble form using an on-column protein refolding system.
  • the protein refolding system according to the present invention is simpler and faster than conventional methods using dialysis or chaperones, thus having useful applications to other inclusion bodies.
  • FIG. 1 shows the expression of stromal cell-derived factor- 1 a (SDF-I a) in a soluble form and an insoluble complex form in an E. coli expression system, wherein lane M shows a marker with a lowest band of 14.4 kDa, lane BF shows a state before IPTG treatment, lanes 2h and 4h show states after treatment with 0.4 mM IPTG for 2 and 4 hours, respectively, lane " soluble” shows proteins existing in an aqueous layer after cell lysis, and lane " insoluble” shows insoluble protein aggregates, obtained after cell lysis, at the point indicated by the arrow.
  • SDF-I a stromal cell-derived factor- 1 a
  • FIG. 2 is a diagram illustrating a protein refolding process wherein denatured
  • SDF-I a contained in a cell lysate is immobilized onto Ni-NTA (nitrilotriacetic acid) resins in a pre-equilibrated HiTrap Chelating HP column, treated with a refolding buffer for 6 - 10 hrs, and washed with 45 mM imidazole buffer, followed by elution with 300 mM imidazole buffer.
  • Ni-NTA nitrilotriacetic acid
  • FIG. 3 is an absorbance profile upon the refolding of SDF-I a on a HiTrap Chelating HP column.
  • FIG. 4 is a photograph taken after the SDS electrophoresis of SDF-I a purified to a purity of 95% or higher, which is detected at 8 kDa as indicated by the arrow, along with a standard protein marker (left lane) with a lowest band detected at 14.4 kDa.
  • FIG. 5 is a graph showing the cell chemotaxis of the refolded SDF-I a according to the present invention in a dose-dependent manner upon the treatment of the human T cell leukemia CCRF-CEM with 10, 50, 100 and 250 nM of SDF-I a, wherein the same results are obtained for migrated cells upon treatment with a mutant (K1A) of SDF-I a and without treatment (Con).
  • K1A mutant of SDF-I a and without treatment
  • a human stromal cell-derived factor 1 a (SDF-I a) gene was amplified from a human fibroblast cDNA library by PCR using a pair of primers (Primer-5' : 5' - GCGGATCCAAGCCCGTCAGCCTGAG-3' , SEQ ID NO.: 1 ; Primer-3' : 5' - CGCTCGAGTTACTTGTTTAAAGCTTTCTCCAG-S' , SEQ ID NO: 2), which are modified from the nucleotide sequence of SDF-I a.
  • the synthesized SDF-I a gene was digested with BamHI and Xhol and jointed into the T7 promoter of pET-28a in the presence of a ligase to construct a recombinant expression vector.
  • the recombinant expression vector constructed in Example 1 was transformed into E. coli.
  • ER2566 cells Novagen
  • ER2566 cells were first inoculated into an LB broth, incubated overnight at 37 2 C and then amplified to an OD 600 of 0.4 in an LB broth. After being harvested by centrifugation, the cells were suspended in 500 ml of sterile deionized water and centrifuged at 2,500 rpm and 4 2 C for 20 min. The cell pellet thus obtained was suspended in 250 ml of 10% glycerol, followed by centrifugation at 4 S C and 2,500 rpm for 20 min.
  • the suspension and centrifugation were repeated, with the exception that 10 ml, instead of 250 ml, of 10% glycerol was used, to afford competent cells.
  • the ER2566 competent cell pellet was suspended in 40 ⁇ I of 10% glycerol to which the constructed recombinant expression vector was then added, followed by transformation using an electroporation method. Thereafter, the cells were incubated in 1 ml of a fresh SOC broth (0.5% yeast extract, 2% tryptophan, 8.5 mM NaCI, 2.5 mM KCI, 20 mM glucose, 10 mM MgCI 2 ) at 37 Q C with shaking at 250 rpm.
  • the cell culture was spread over an LB plate containing 50 ⁇ g/ml of kanamycin to select transformants anchoring wild-type SDF-I a or mutants thereof.
  • the pET-28a carrying an SDF-I a gene was transformed into E. coli BL21 (DE3).
  • a mutant SDF-I a (K1 A) gene was synthesized through polymerase chain reaction using site-directed mutagenesis. The mutant gene was transformed into E. coli BL21 (DE3) in the same manner as described above.
  • SDF-I a gene prepared in Example 1 was cultured at 37 2 C to the degree of an OD 600 of 0.6 in 2 liters of LB broth, after which wild-type and mutant proteins were induced in the presence of 0.4 mM at 37 S C express them. After 4 hours of the induction, the cells were harvested by centrifugation and resuspended in 100 ml of a buffer (10 mM sodium phosphate, 150 mM NaCI, pH7.4) containing a protease inhibitor. To the suspension was added lysozyme (1 .0 mg/ml) to destroy cell membranes, followed by sonication to rupture cells.
  • the insoluble complex obtained by centrifuging the cell lysate thus obtained was subjected to solubilization with a denaturing buffer (8 M urea, 20 mM Tris-HCI, pH 8.0, 500 mM NaCI, 0.5 mM b-mercaptoethanol, 3% glycerol) for 2 hrs at room temperature. Centrifugation at 15,000 rpm for 30 min afforded a denatured extract.
  • a denaturing buffer 8 M urea, 20 mM Tris-HCI, pH 8.0, 500 mM NaCI, 0.5 mM b-mercaptoethanol, 3% glycerol
  • Example 2 The extract obtained in Example 2 was applied to the HiTrap Chelating HP column (GE Healthcare) pre-equilibrated with Ni ions and a denaturant buffer. After the extract was loaded at a rate of 3 ml per min on the column, a refolding solution was slowly poured at a rate of 0.2 ml per min for 6 - 10 hrs to replace the pre- existing buffer therewith.
  • HiTrap Chelating HP column GE Healthcare
  • the SDF-I a prepared in Example 3 was assayed for cell chemotaxis using the human leukemia cell line CCRF-CEM on which CXCR4, a receptor of SDF-I a, is expressed, commercially available from the Korean Cell Line Bank (KCLB).
  • the CCRF-CEM cell line was maintained in an RPMI 1640 medium supplemented with 25 mM Hepes and 10% FBS.
  • the CCRF-CEM cells were plated at a density of 2x10 6 cells/ml and washed with an assay solution (RPMI 1640, 25 mM Hepes, pH 7.5, 0.1 % BSA).
  • the chemotactic ability of SDF-I a was analyzed using a 48-well ChemoTx chamber (NeuroProbe, Gaithersburg, MD) in which 27 ⁇ I of the SDF-I a was placed in each of the lower wells while 50 ⁇ I of CCRF-CEM cells was placed in each of the upper wells. After the chamber was incubated at 37 S C for 3 hrs in a 5% CO2 incubator, the CCRF-CEM cells that migrated into the lower wells were counted to determine cell chemotaxis, compared with that of the wild-type SDF-I a or the mutant (K1 A).

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Abstract

L'invention porte sur un procédé de production d'une forme active du facteur humain issu des cellules stromales (SDF-1 a), comportant la renaturation d'un corps d'inclusion de protéines exprimées dans une grande quantité dans E. coli dans une forme active à l'aide d'une colonne. Egalement, l'invention porte sur un SDF-1a solubilisé actif. Le système de repliement de protéine est plus simple et plus rapide que les procédés classiques utilisant la dialyse ou les chaperons, permettant ainsi des applications utiles à d'autres corps d'inclusion.
PCT/KR2008/000265 2007-07-03 2008-01-16 Procédé de fabrication du facteur 1a issu de cellules stromales comme forme de protéine soluble active Ceased WO2009005201A1 (fr)

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KR1020070066489A KR20090003652A (ko) 2007-07-03 2007-07-03 인간 기질 세포 유도체의 수용성 단백질로의 재생 방법
KR10-2007-0066489 2007-07-03

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CN106243186B (zh) * 2015-06-15 2020-12-25 张鹏 可独立用于蛋白质复性或作为蛋白质复性前导操作的循环运作方法

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KR100409092B1 (ko) * 2000-12-22 2003-12-11 주)녹십자 단백질의 제조 방법
KR100482190B1 (ko) * 2000-02-17 2005-04-13 주식회사 엘지생명과학 대장균을 이용한 삼일열 말라리아 원충의 표면 단백질의 제조방법

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VELDKAMP C.T. ET AL.: "On-column Refolding of Recombinant Chemokines for NMR Studies and Biological Assay", PROTEIN EXPERIMENTAL PURIFICATION, vol. 52, no. 1, March 2007 (2007-03-01), pages 202 - 209, XP005758624 *

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