WO2011131491A1 - Procédé de production d'une protéine recombinante soluble - Google Patents

Procédé de production d'une protéine recombinante soluble Download PDF

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WO2011131491A1
WO2011131491A1 PCT/EP2011/055423 EP2011055423W WO2011131491A1 WO 2011131491 A1 WO2011131491 A1 WO 2011131491A1 EP 2011055423 W EP2011055423 W EP 2011055423W WO 2011131491 A1 WO2011131491 A1 WO 2011131491A1
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protein
recombinant protein
adiponectin
recombinant
gad
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PCT/EP2011/055423
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English (en)
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John Heiker
Annette G. Beck-Sickinger
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Scil Proteins Gmbh
Universitaet Leipzig
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Publication of WO2011131491A1 publication Critical patent/WO2011131491A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • 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
    • C07K14/525—Tumour necrosis factor [TNF]
    • 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
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat

Definitions

  • the present invention is directed to a method of producing a soluble recombinant protein by in vitro folding.
  • the present invention is further directed to a recombinant protein, obtainable by this method as well as to a pharmaceutical composition comprising recombinant adiponectin or adiponectin-like protein for use in the treatment of obesity-related metabolic disorders.
  • inclusion bodies When foreign genes are expressed in another host, for example in a bacterial host, the resulting proteins sometimes are forming so called inclusion bodies. This is in particular true when large evolutionary distances are crossed, for example, if a cDNA isolated from human beings is expressed as a recombinant gene in a prokaryote cell. It is estimated that 70% - 80% of the proteins, expressed for example in E. coli, by recombinant techniques are contained in inclusion bodies (i.e. as protein aggregates). These inclusion bodies are nuclear or cytoplasmic aggregates of proteins and typically represent sites of viral multiplication in a bacterium and usually consist of viral capsid proteins.
  • the purification of the expressed proteins from inclusion bodies usually requires two main steps, i.e. the extraction of inclusion bodies from the bacteria followed by the solubilisation of the purified inclusion bodies and recovery of the refolded native protein. This is considered labor-intensive, time consuming and not very cost-effective. Therefore, one major drawback of the use of proteins, such as their therapeutic application, remains the lack of an easy, cost- efficient production of functional protein.
  • the present invention provides an improved method of producing soluble recombinant proteins by in vitro folding, which makes use of the so called alkaline-shock solubilisation method in order to solubilise inclusion body proteins.
  • the application of this method for the production of recombinant proteins leads to high level production of proteins from bacterial inclusion bodies without the necessity of using chaotropic agents in the process.
  • the methods of the present invention involve an alkaline-shock solubilisation step followed by precipitation of the readily renaturing protein.
  • Precipitation of the mildly solubilised protein capitalises on the advantages of inclusion body formation and benefits from the present secondary-like structure in inclusion body proteins.
  • This approach to the crucial steps in inclusion body protein recovery provides access to gram scale amounts of recombinant proteins with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralise the pH and to renature the protein, thus saving chemicals and time.
  • the precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in vivo. Using fat-batch fermenters in industrial expression systems this economic process will provide large-scale yields of recombinant protein facilitating the use of it in therapeutic application.
  • the invention is directed to a method of producing a soluble recombinant protein by in vitro folding comprising the steps of:
  • bacterial host cells such as E. coli or Bacillus subtilis.
  • a preferred host cell is E. coli strain B121(DE3)pLysS_RARE.
  • Further bacterial host cells which could be used in the present invention, comprise Streptococci, Staphylococci, Streptomyces cells, among others.
  • the first step of producing soluble recombinant protein according to the invention is to provide the nucleic acids coding for the respective protein. These nucleic acids are cloned into a usual expression vector comprising one or more regulatory sequences.
  • expression vector generally refers to a plasmid or phage or virus or vector for expressing a polypeptide from a DNA (RNA) sequence.
  • An expression vector can comprise a transcriptional unit comprise an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, (3) appropriate transcription initiation and termination sequences.
  • the recombinant construct will be introduced into the host cell by well-known methods such as calcium phosphate transfection, DEAE, dextran mediated transfection, rubidium chloride method or electro poration (Davis, L. et al., Basic Methods in Molecular Biology (1986)). Basically, appropriate cloning and expression vectors for use with prokaryotic host cells and the corresponding methods are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, New York (1989).
  • a preferred example of an expression vector is the pET-15b bacterial expression vector.
  • the inclusion bodies containing the recombinant protein are isolated and solubilised in a suitable solvent by adding an alkaline agent.
  • this alkaline agent is not restricted, it is preferred to select the alkaline agent from NaOH, KOH or NH 4 OH in a suitable concentration.
  • suitable concentration refers to such an amount of alkaline agent to be added to the solution containing the isolated inclusion bodies leading to an adjustment of the pH of the solution to 12 - 13, preferably 12.5.
  • adjusting the pH of the solution to 12.5 using 1 M NaOH results in complete solubilisation of the inclusion bodies within seconds.
  • solution as used herein should be understood broadly and also encompasses liquid systems such as emulsions or suspensions.
  • the suitable solvent used in step b) preferably is selected from a buffer solution, such as TRIS, hepes or phosphate buffer.
  • a buffer solution such as TRIS, hepes or phosphate buffer.
  • TRIS hepes or phosphate buffer
  • 20 mM TRIS buffer pH 8 before the alkaline agent is added.
  • the inclusion bodies usually are resuspended in the buffer leading to a suspension (i.e. not a clear solution).
  • a clear solution is, however, achieved shortly after adding the alkaline agent in a suitable amount.
  • the recombinant protein is precipitated and isolated from the solution by adding an organic solvent.
  • This organic solvent is such that it must guarantee precipitation of the recombinant protein form the solution. That is to say, the solubility of the proteins in the aqueous buffer depends on the distribution of hydrophilic and hydrophobic amino acid residues on the protein surface. Hydrophobic residues predominantly occur in the globular protein core, but some exist in patches on the surface. As it is known to a skilled person, the addition of miscible solvents such as ethanol or methanol to a solution may cause proteins in the solution to precipitate.
  • the solvation layer around the protein will decrease as the organic solvent progressively displaces water from the protein surface and binds it in hydration layers around the organic solvent and molecules.
  • Important parameters to consider are the temperature, which should be appropriate to avoid denaturation, the pH and the protein concentration in the solution.
  • organic solvents In the present invention, all conceivable kinds of organic solvents might be used, but the solvents should be miscible with the solvent used in step b) of the method of the present invention.
  • acetone it is particular preferred to use acetone as an organic solvent, however, other organic solvents such as ethanol or methanol could be used as well.
  • the method of producing a soluble recombinant protein of the present invention involves an immediate precipitation of the soluble protein after adding the organic solvent. This is to avoid an irreversible modification of the protein due to the extreme basic pH between 12 and 13. Or, in other words, steps b) and c) of the method of the present invention should be performed shortly after each other.
  • "Shortly" in this context means a time frame of a one second to a view minutes, for example not more than 5 minutes.
  • the protein pellet After centrifugation and washing with acetone or another suitable organic solvent, the protein pellet is ready soluble in water or buffer in suitable concentrations.
  • the recombinant protein is selected from human proteins, fusion proteins and fragments thereof, each containing beta pleated sheet structures.
  • This beta pleated sheet structure is believed to be the preferred structural element of protein aggregates and, thus, a common denominator in protein aggregation.
  • the combination of mild alkaline-shock solubilisation and immediate precipitation of the protein bypasses the need for large volumes of cost-prohibitive refolding buffers and expensive chemicals and additives required for most widely used dilution-based refolding strategies, thus saving time and money.
  • the precipitated protein can be dissolved/renaturated at very high concentrations (100 mg/mL) making protein sample preparation for follow up chromatography steps or endotoxin removal very convenient.
  • the recombinant protein of the invention is selected from adiponectin or adiponectin-like proteins.
  • the adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects.
  • Adiponectin levels are in the microgram per mL range in healthy humans and inversely correlated to obesity and metabolic disorders. Based on these properties, raising the blood plasma level of adiponectin by direct administration is an intriguing therapeutic strategy. Obtaining large amounts of recombinant protein is a primary obstacle to therapeutic application.
  • Adiponectin is one of the most abundant adipokines and since the independent identification of the protein by four research groups in the mid nineties a growing body of evidence represents adiponectin as a key player in regulation of insulin sensitivity, inflammation and energy homeostasis 1 3 .
  • adiponectin plasma levels are inversely correlated to obesity and its related disorders as type 2 diabetes, hepatic dysfunction, atherosclerosis and cardiovascular disease. Taken together these results make adiponectin a promising candidate for drug development and treatment of obesity-related metabolic disorders.
  • Full-length adiponectin forms a wide range of complexes from low, medium to high molecular weight complexes (LMW, MMW and HMW) and appears to be involved in different and tissue specific signalling pathways.
  • the proteolytic globular fragment (gAd) is present at significantly lower concentrations in the plasma, but shows more potent actions at lower concentrations compared to the full length protein in animal studies 3 ' 4 .
  • AdipoRl adiponectin receptor 1
  • AdipoR2 adiponectin receptor 2
  • AdipoRl shows high affinity for gAd and a reduced affinity for fAd
  • AdipoR2 exerts medium affinity for both variants.
  • the receptors contain seven transmembrane domains but are structurally and functionally distinct from G-protein coupled receptors (GPCRs).
  • GPCRs G-protein coupled receptors
  • bacterially expressed recombinant gAd has shown to exert great therapeutic potential.
  • Fruebis et al demonstrated increased oxidation in muscle tissue after acute gAd treatment which resulted in sustainable weight loss in high fat/sucrose fed mice 2 .
  • gAd expressed in yeast significantly lowered blood glucose levels in STZ- induced type 1 diabetic mice and promoted free fatty acid clearance in hyper lipemic mice 5 .
  • Recently the first chronic treatment with the trimeric fraction of recombinant gAd improved glucose tolerance and totally reversed insulin resistance in high-fat diet induced diabetic mice
  • the adiponectin- like proteins is selected from the group consisting of Clq/TNF related proteins.
  • Clq/TNF related proteins it is referred to R. Ghai et al., Immunobiology 212 (2007) 253 - 266, discussing C l q and its growing family.
  • the adiponectin- like proteins selected from Clq and tumor necrosis factor related proteins 1 - 9 are disclosed in table 1 of this paper.
  • the present invention is directed to recombinant protein, obtainable by the methods outlined above.
  • This recombinant protein for example adiponectin or adiponectin-like protein, might be comprised in a pharmaceutical composition further containing a suitable pharmaceutical acceptable carrier or diluent.
  • a suitable pharmaceutical acceptable carrier or diluent may contain diluents, fillers, salts, buffers, stabilisers, solubilisers and other materials well known in the art.
  • pharmaceutically acceptable means a nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s).
  • the characteristics of the carrier will depend on the route of administration.
  • the composition may further contain other agents which either enhance the activity or use in treatment. Such additional factors and/or agents may be included in the composition to produce a synergistic effect or to minimise side-effects.
  • compositions contain a therapeutically effective dose of the recombinant protein.
  • a therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of pathophysiological conditions.
  • Suitable routes of administration may, for example, include oral and parenteral delivery, including intramuscular and subcutaneous injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal injections.
  • this pharmaceutical composition might be used in the treatment of obesity-related metabolic disorders.
  • Figure 1 Alkaline- shock solubilises gAd from inclusion bodies and yields recombinant protein of great purity.
  • A SDS-PAGE analytic of different stages of gAd inclusion body preparation and solubilisation as the coomassie stain of the lysis supernatant (lane 1), inclusion body washings (lane 2-4), urea (lane 5-7) and alkaline-shock solubilisations (lane 8).
  • B Coomassie and silver stain of acetone precipitated gAd solved in water (1 ⁇ g per lane and 200 ng per lane, respectively).
  • Figure 2 Circular dichroism spectrum of alkaline-shock solubilised gAd at 5 ⁇ in water. CD data are presented as mean residue weight ellipticity. Insertion shows the PDB rendering of murine gAd homotrimer based on 1C28.
  • FIG. 3 Induction of ACC phosphorylation (Ser79) in MCF7 cells by alkaline-shock solubilised gAd.
  • MCF7 cells were serum starved (5 h) and treated with globular adiponectin (5 ⁇ g/ml) for 5 min.
  • Cell lysates were separated by SDS-PAGE, subjected to immunoblotting using anti-ACC and anti-PACC antibodies .
  • the graphic representation of ACC phosphorylation is shown in (A), respective Western Blot bands in (B). Data shown represent the means ⁇ s.e.m. of three experiments performed. * statistically different from control with P ⁇ 0.01.
  • FIG. 4 Blood glucose lowering in STZ induced TID mice by alkaline- shock solubilised gAd.
  • STZ induced type 1 diabetic mice with blood glucose levels > 17 mmol/L were injected gAd (5 mg/kg BW, i.p.) or pyrogen free saline and 4 h after treatment blood glucose levels were determined.
  • Globular adiponectin was cloned into pET-15b bacterial expression vector.
  • Protease inhibitor cocktail was from Sigma- Aldrich.
  • ACC and phospho-ACC specific antibodies were from Cell Signalling Technologies (Danver, MA).
  • Escherichia coli strain DH5a was used as a host for plasmid constructions and E. coli BL21(DE3)pLysS RARE was used for bacterial expression of His 6 -tagged protein.
  • E. coli B121(DE3)plysS_RARE were transformed with the plasmid gAd_pET15b and grown on solid LB/ampicillin (100 ⁇ g/mL) plates at 37°C overnight. A single colony was selected to grow a 50 mL starter culture overnight at 37°C. The starter culture was centrifuged at 1500g for 5 min and the cell pellet was resuspended in 10 mL LB. The starter culture was inoculated in 1.5 L LB/ampicillin (100 ⁇ g/mL) and incubated at 37°C with shaking until the OD 6 oo reached 0.6.
  • the pellet ( ⁇ 3 g wet pellet) was resuspended in ⁇ 5 volumes of buffer containing 25 mM TRIS, 500 mM NaCl, 2 M urea, 0.25% TritonX-100, pH 8.0, incubated for 1 h at RT and centrifuged at 18000g for 45 min at 4°C. This was repeated twice and a third and fourth time without Tritonx-100 to remove detergent. The washed pellet was subjected to three solubilisation steps.
  • Inclusion bodies were resuspended in buffer (20 mM TRIS, 500 mM NaCl, 8 M urea, 5 mM ⁇ -mercaptoethanole, pH 8), incubated for 1 h at RT and centrifuged at 18000g for 45 min at 4°C.
  • the remaining pellet (1 g wet pellet) was resupended in 15 mL of buffer (20 mM TRIS, pH 8) and the pH was adjusted to 12.5 using 1 M NaOH resulting in complete solubilisation of the inclusion bodies within seconds.
  • the clear solution was centrifuged for 15 min at 18000g and the supernatant was filtered through a 0.45 ⁇ sterile filter. Protein was precipitated by addition of ⁇ 3 volumes of acetone and precipitated protein was collected by centrifugation at 18000g for 15 min. The pellet was washed once with acetone, spun down and air dried for storage at -70°C.
  • Precipitated protein readily dissolved in distillated water or buffer (10 mM Tris, 150 mM NaCl, pH 7.5) up to concentrations of 100 mg/rnL. Aliquots were stored at - 70°C or lyophilized. Endotoxin removal for recombinant gAd used in vivo experiments was done using an ActiClean Etox column (Sterogene) following the manufacturers' instructions.
  • Recombinant protein was analysed by MALDI-TOF mass spectrometry using a MALDI- TOF/TOF UltraflexIII (Bruker Daltonics) in linear mode. Spectra were calibrated using a mixture of recombinant cytochrome c and myoglobin from horse heart (both Fluka) as standard. Tryptic in solution digests were analyzed by MALDI-TOF MS and MSMS. Peak lists of the tryptic peptide masses were generated and searched against the National Center for Biotechnology Information non-redundant database using the Mascot search engine (Matrix Science, London, UK; http://www.matrixscience.com) in order to identify the proteins.
  • CD spectra were recorded using a JASCO model J720 spectropolarimeter over 250-185 nm at 20°C in a N 2 atmosphere. Protein solution was measured at a concentration of 5 ⁇ in water. Each measurement was repeated 3 times using a thermo stable sample cell with a path of 0.2 cm and following parameters: response time of 4 s, scan speed of 20 nm/min, sensitivity of 10 mdeg, step resolution of 0.2 nm, and bandwidth of 2 nm.
  • the CD spectrum of the solvent was subtracted from the CD spectra of the protein solutions to eliminate the interference from solvent and optical equipment. High-frequency noise was reduced by means of a low-path Fourier transform filter. The ellipticity was expressed as the mean residue weight ellipticity [0]NRW in deg cm 2 dmol "1 .
  • MCF7 cells were grown in DMEM Ham's F-12 medium (PAA) supplemented with 10% FCS. Cells were serum starved for 5 h before stimulation experiments. After treatment with 5 ⁇ g/mL gAd in DMEM Ham's F12 for 5 min cells were scraped into ice-cold lysis buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, protease inhibitor cocktail, pH 7.5) and sonicated with 15 pulses on ice (20 % power, 0.5 amplitude). The centrifuged ly sates were subjected to SDS-PAGE and Western Blot analysis.
  • PPA DMEM Ham's F-12 medium
  • Recombinant gAd is expressed as inclusion bodies and prone to urea solubilisation
  • the urea solubilised fraction contained major impurities and less than 50% gAd (Fig. 1A).
  • the main host impurity was identified as E. coli 16 kDa heat shock protein A according to Mascot peptide mass fingerprint analysis following in gel tryptic digestion and MALDI-TOF MS (data not shown).
  • the air dried protein pellet ( ⁇ 1 g / L culture medium) is readily soluble in water or buffer at concentrations up to 100 mg/mL. SDS-PAGE followed by Coomassie and silver stain showed a purity of the protein >95 % (Fig. I B) and MALDI-TOF and Mascot peptide mass fingerprint analysis after in solution tryptic digestion and MALDI-TOF MS and MSMS proved protein identity as His-tag human gAd fusion protein (Fig 1C). Next the inventors further performed size exclusion chromatography to investigate possible aggregation of the pH solubilised gAd at high protein concentration (-100 mg/mL) which showed only very little aggregated protein ( ⁇ 5 %) (Fig. 2A).
  • alkaline-shock solubilisation in combination with acetone precipitation of inclusion body derived gAd led to water or buffer soluble, non aggregated and properly folded recombinant protein with gram yields per litre culture medium.
  • Recombinant gAd induces ACC-phosphorylation in MCF7 cells
  • Adiponectin has enjoyed immense attention over the past years as it has emerged as a potent insulin sensitizing protein as well as a protector against cardiovascular disease with beneficial effects on many parameters associated with the metabolic syndrome.
  • adiponectin as a reliable biomarker for these disorders and an important element of metabolic improvement thus representing an interesting therapeutic agent, but recombinant expression of large amounts of active adiponectin remains the bottleneck for therapeutic application.
  • the inventors managed to express globular adiponectin from E. coli in large amounts to overcome this problem.
  • adiponectin Recombinant expression of adiponectin in the literature was performed mainly in E. coli or in the eucaryotic system of HEK 293 cells 4 ' 7"9 .
  • Bacterial recombinant adiponectin was obtained as inclusion bodies or as soluble protein 7 ' 10 .
  • Liu and Liu et al published the expression gAd in Pichia pastoris with yields of about 50 mg/L culture medium 5 .
  • inclusion bodies due to overexpression in E. coli may present a disadvantage at first sight yielding in principle inactive protein with the need for solublilisation and subsequent refolding to recover the native protein.
  • a standard solubilisation procedure using 8 M urea only a minor fraction of gAd, but a major fraction of host cell impurities are solubilised (Fig 1 A), so we incorporated the 8 M urea solubilisation into the inclusion body washing procedure to achieve near homogenity before solubilisation.
  • a further advantage is a lower degradation risk with the inclusion body state protecting against proteolysis by host proteases.
  • the homogenity in inclusion bodies is generally high reducing the need for subsequent purification steps.
  • the inventors recovered gAd by a chaotropeless alkaline- shock solubilisation of the rigorously washed inclusion bodies in Tris buffer followed by immediate acetone protein precipitation to prevent protein modification due to the extreme pH.
  • This mild solubilisation method preserves native like secondary structure of the protein which after precipitation readily renatures in water or Tris buffer (pH 7.5).
  • the gAd structure consists of solely B- sheets representing the preferred structural element in protein aggregates and intermolecular sheet-formation is believed to be a common denominator in protein aggregation.
  • the present protocol could be shown to further facilitate the easy renaturation of the recombinant gAd.
  • the complete solubilisation of inclusion bodies in least amounts of buffer compared to insolubility at neutral pH suggests that the charge distribution at alkaline pH across the polypeptide facilitates the solubilisation of gAd. Hydrophobic interactions seem to be of neglectable effect in the process resulting in complete solubilisation without the addition of even low chaotropic agent concentration. This suggests ionic interactions as the main contribution to gAd aggregation as inclusion bodies.
  • the combination of mild alkaline-shock solubilisation and immediate precipitation of the protein bypasses the need for large volumes of cost-prohibitive refolding buffers and expensive chemicals and additives required for most widely used dilution-based refolding strategies, thus saving time and money.
  • the precipitated protein can be dissolved/renatured at very high concentrations (100 mg/mL) making protein sample preparation for follow up chromatography steps or endotoxin removal very convenient.

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Abstract

La présente invention concerne un procédé de production d'une protéine recombinante soluble par repliement in vitro. La présente invention concerne également une protéine recombinante, pouvant être obtenue par ce procédé, ainsi qu'une composition pharmaceutique comprenant de l'adiponectine recombinante ou une protéine analogue à l'adiponectine recombinante en vue d'une utilisation dans le traitement de troubles métaboliques se rapportant à l'obésité.
PCT/EP2011/055423 2010-04-22 2011-04-07 Procédé de production d'une protéine recombinante soluble WO2011131491A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN113683676A (zh) * 2021-08-27 2021-11-23 恒敬合创生物医药(上海)有限公司 一种环保的可逆的蛋白变性工艺

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030166062A1 (en) * 2001-02-23 2003-09-04 Gonzalez-Villasenor Lucia Irene Methods and compositions for production of recombinant peptides
WO2009045553A1 (fr) * 2007-10-05 2009-04-09 Barofold, Inc. Traitement sous haute pression d'interférons agrégés

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030166062A1 (en) * 2001-02-23 2003-09-04 Gonzalez-Villasenor Lucia Irene Methods and compositions for production of recombinant peptides
WO2009045553A1 (fr) * 2007-10-05 2009-04-09 Barofold, Inc. Traitement sous haute pression d'interférons agrégés

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HALUZIK M. ET AL: "Adiponectin and its role in the obesity-induced insulin resistance and related complications", PHYSIOL. RES., vol. 53, 1 January 2004 (2004-01-01), pages 123 - 129, XP002648750 *
HEIKER J. ET AL.: "access to gram scale amounts of functional globular adiponectin from E.coli inclusion bodies by alkaline-shock solubilization", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 398, 10 June 2010 (2010-06-10), pages 32 - 37, XP002648801 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113683676A (zh) * 2021-08-27 2021-11-23 恒敬合创生物医药(上海)有限公司 一种环保的可逆的蛋白变性工艺

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