WO2011131491A1

WO2011131491A1 - Method of producing soluble recombinant protein

Info

Publication number: WO2011131491A1
Application number: PCT/EP2011/055423
Authority: WO
Inventors: John Heiker; Annette G. Beck-Sickinger
Original assignee: Universitaet Leipzig; Scil Proteins GmbH
Current assignee: Universitaet Leipzig; Navigo Proteins GmbH
Priority date: 2010-04-22
Filing date: 2011-04-07
Publication date: 2011-10-27
Anticipated expiration: 2012-10-22

Abstract

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.

Description

METHOD OF PRODUCING SOLUBLE RECOMBINANT PROTEIN

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.

BACKGROUND OF THE INVENTION

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.

In the nineteen nineties several groups reported pH assisted solubilisation of inclusion bodies. P.J. Tichy et al, Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies, Protein Expr Purif 4 (1993) 59-63, and Z. Zhang et al, Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH, Sci China C Life Sci 40 (1997) 169-175, report such a pH assisted solubilisation of inclusion bodies. However, the alkaline pH assisted procedures disclosed therein still demand high chaotropic agent concentrations (8 M urea) in order to work properly and complex and time consuming refolding procedures are needed to recover the native protein.

SUMMARY OF THE INVENTION

Therefore, it is the objective problem underlying the invention to provide a method of producing soluble recombinant proteins by in vitro folding which allows an easy, cost- efficient production of functional protein in high yield. It is a further object of the invention to provide such a method, which avoids the use of chaotropic agents.

This is achieved by the subject-matter of the independent claims. Preferred embodiments are described in the dependent claims.

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.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect, the invention is directed to a method of producing a soluble recombinant protein by in vitro folding comprising the steps of:

a) expressing the recombinant protein in a bacterial host and isolating inclusion bodies of said recombinant protein;

b) solubilising the inclusion bodies in a suitable solvent by adding an alkaline agent; c) precipitating and isolating the recombinant protein from the solution by adding an organic solvent.

As a host cell, in particular bacterial host cells may be used such as E. coli or Bacillus subtilis. A preferred host cell is E. coli strain B121(DE3)pLysS_RARE. However, it should be clear that the invention will also work with other kinds of prokaryotic host cells. 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. The term "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.

Once the inclusion bodies containing the recombinant protein have been formed in the bacterial host cell, they are isolated and solubilised in a suitable solvent by adding an alkaline agent. Although the selection of this alkaline agent according to the invention is not restricted, it is preferred to select the alkaline agent from NaOH, KOH or NH₄OH in a suitable concentration.

The term "suitable concentration" as used above 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. For example, adjusting the pH of the solution to 12.5 using 1 M NaOH results in complete solubilisation of the inclusion bodies within seconds.

The term "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. For example, it is appropriate to use 20 mM TRIS buffer pH 8 before the alkaline agent is added. 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.

In a final step, the recombinant protein is precipitated and isolated from the solution by adding an organic solvent. The nature of 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.

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. Here, 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.

In a preferred embodiment, 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.

After centrifugation and washing with acetone or another suitable organic solvent, the protein pellet is ready soluble in water or buffer in suitable concentrations.

According to a preferred embodiment, 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.

As noted above, in the 1990's several groups reported pH assisted solubilisation of inclusion bodies. While some alkaline pH assisted procedures still demanded high chaotropic agent concentrations (8 M urea), a mild solubilisation strategy employing only 2 M urea or guanidine hydrochloride at pH > 12 has been applied for the recovery of human growth hormone (HGH) and zona pellucida protein. This mild solubilisation process without disturbing the native-like secondary structure significantly increased the recovery of bioactive HGH via pulsed renaturation process, simple dilution or dialysis. In contrast to the present invention, however, all procedures require solubilisation times of at least 30 minutes to one hour increasing the possibility of irreversible modifications of the protein.

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. Furthermore, 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.

According to a further preferred embodiment, 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.

Adipose tissue as an endocrine organ secretes a wide variety of adipokines. 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}. Although mainly secreted by adipose tissue 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 (fAd) 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.

Two receptors have been described as adiponectin receptor 1 (AdipoRl) and adiponectin receptor 2 (AdipoR2). AdipoRl shows high affinity for gAd and a reduced affinity for fAd whereas 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). We recently confirmed the inverse membrane orientation of AdipoRs in the plasma membrane compared to GPCRs with an intracellular N-terminus and an extracellular C-terminus and reported casein kinase 2 as interaction partner of the AdipoRl .

Despite the ongoing discussion on which form of recombinant adiponectin, HMW, MMW or LMW form, is to be administered, 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. Also 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

6

In another embodiment, the adiponectin- like proteins is selected from the group consisting of Clq/TNF related proteins. In this connection, it is referred to R. Ghai et al., Immunobiology 212 (2007) 253 - 266, discussing C l q and its growing family. As an example, the adiponectin- like proteins selected from Clq and tumor necrosis factor related proteins 1 - 9 are disclosed in table 1 of this paper.

In a second aspect, 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. Such a composition may contain diluents, fillers, salts, buffers, stabilisers, solubilisers and other materials well known in the art. The term "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.

Techniques for formulation and administration of the compounds of the present application may be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition.

The 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.

Furthermore, this pharmaceutical composition might be used in the treatment of obesity- related metabolic disorders.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The invention is now further illustrated by Examples and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

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 μg per lane and 200 ng per lane, respectively). (C) MALDI-TOF spectrum of gAd in linear mode([M+H]⁺¹t eoreticai=18069. Together with the singly charged ion a doubly and triply charged ion is observed

Figure 2: Circular dichroism spectrum of alkaline-shock solubilised gAd at 5μΜ in water. CD data are presented as mean residue weight ellipticity. Insertion shows the PDB rendering of murine gAd homotrimer based on 1C28.

Figure 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 μg/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.

Figure 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. EXAMPLES

Material and methods

Plasmids, antibodies and chemicals.

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).

Bacterial strains.

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 His₆ -tagged protein.

Protein expression.

E. coli B121(DE3)plysS_RARE were transformed with the plasmid gAd_pET15b and grown on solid LB/ampicillin (100 μg/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 μg/mL) and incubated at 37°C with shaking until the OD₆oo reached 0.6. Expression of recombinant gAd was induced by addition of IPTG to a final concentration of 1 mM and growth was continued for 6 h at 37°C. Finally cells were harvested and collected by centrifugation at lOOOOg for 10 min. The cell pellet was resuspended in lysis buffer (25 mM TRIS, 500 mM NaCl, 5 mM MgCl₂, pH 8 containing lyophilized Dnasel) and lysis was performed in 5 freeze and thaw cycles using liquid nitrogen and a 30°C water bath. Bacterial lysates were centrifuged at 18000g at 4°C for 45 min and the pellet was stored at -70°C. Inclusion body treatment

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 β-mercaptoethanole, pH 8), incubated for 1 h at RT and centrifuged at 18000g for 45 min at 4°C.

Alkaline-shock solubilisation and subsequent protein precipitation of pure recombinant gAd

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 μιη 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.

SDS-PAGE and Western Blot

Samples were run on 15% SDS-gels and stained with coomassie Brilliant Blue R250 (Sigma- Aldrich), silver stained (Fermentas). 7% SDS-Gels of cell lysates (30 μg per lane) were transferred onto PVDF-membrane (HyPond, GE Healthcare), blocked with protein free blocking solution (Pierce) for 1 h at RT and incubated with primary antibody for 1 h at RT or overnight at 4°C. Blots were washed with TBST (3 x 5 min), incubated with horseradish- peroxidase coupled secondary antibody for 1.5 h at RT and developed using ECL-substrate (Pierce) and G:BOX ChemieXL camera (Syngene). Western Blot bands were quantified using GeneSnap software (Syngene) and values are presented as the mean ± s.e.m. of at least three experiments performed. Statistical evaluation of the data was done using one-way analysis of variance (ANOVA) with the Graph-Pad Prism program (GraphPadlnc). Statistical significance is indicated as follows: *(P<0.05).

Protein concentration determination

Protein concentrations were determined with Bio-Rad Protein Assay (Bio-Rad), using BSA as a standard.

MALDI-TOF/TOF mass spectrometry

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. For database searches the following parameters were used - species: Homo sapiens, tryptic digestion with a maximum of one missed cleavage site, monoisotopic masses; variable modification: methionine residues oxidized; mass tolerance MS : 50 ppm; mass tolerance MSMS: 0.25 Da.

Size exclusion chromatography

Aggregation state of purified gAd was addressed by size exclusion chromatography on a Superdex S200 26/60pg column with 10 mM Tris, 150 mM NaCl, pH 7.5 as running buffer using the AKTA-purifier system (GE Healthcare). Circular dichroism spectroscopy

CD spectra were recorded using a JASCO model J720 spectropolarimeter over 250-185 nm at 20°C in a N₂ atmosphere. Protein solution was measured at a concentration of 5 μΜ 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 cm² dmol^"1.

Cell culture and activity assay

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 μg/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.

In vivo experiments

Pathogen free 12 week old male C57BL6/J mice were maintained under controlled animal care conditions with free access to standard chow and water. Mice were given two i.p. injections of streptozocin (STZ) (150mg/kg BW) after fasting for 14h. Two weeks later, the glucose levels were measured by Hitado Analyzer (Mohnesee, Germany) after fasting overnight. Those mice with glucose levels > 17mmol/l were considered as type 1 diabetic and divided into 2 groups (N=4 each group). They were injected (i.p.) with 5mg/kg BW of recombinant gAd or pyrogen- free saline respectively. After 4 hour of treatment blood glucose levels were analyzed. All experiments were performed in accordance with the rules for animal care of the local government authorities (Landesdirektion Leipzig, Germany) and were approved by the institutional animal care committee. Results:

Recombinant gAd is expressed as inclusion bodies and prone to urea solubilisation

After expression for 6 h at 37 °C cells were centrifuged and lysed. SDS-Page analysis revealed expression as inclusion bodies. After inclusion body washing steps and three solubilisation steps using 8 M urea about one third of the pellet remained unsolubilised. SDS- PAGE analysis showed inefficient solubilisation of further protein. 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). Purification and on column refolding of urea-solubilised gAd using the AKTA-purifier chromatography system yielded only low mg-amounts of recombinant protein per litre of expression culture (data not shown). SDS-PAGE analysis of the residual pellet revealed the vast majority of the recombinant gAd with high purity in the urea insoluble pellet (Fig. 1 A).

Alkaline-shock solubilisation of gAd inclusion bodies yields pure, non aggregated and properly folded gAd in gram-scale

The pH-assisted solubilisation of inclusion bodies with caltrops, such as urea or guanidine hydrochloride, is reported in the literature. After first tests using urea and pH above 12, we tried to reduce the urea concentration needed for pH-assisted solubilisation and found a simple Tris buffer adjusted to pH 12.5 as an alkaline-shock sufficient for the solubilisation of the chaotrope insoluble pellet within seconds. To avoid irreversible modification of our protein due to the extreme basic pH the soluble protein was immediately precipitated by the addition of 3 volumes of ice cold acetone. After centrifugation and washing with acetone 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).

The fact that the acetone precipitated protein is readily soluble in water already gave indications for a properly folded protein. Circular dichroism studies confirmed this, resulting in spectra consistent with a negative cotton effect at 218 nm and a positive cotton effect at 192 nm. This is typical for a fully β-sheet folded structure and in agreement with the crystal structure solved for murine gAd (Fig. 2). The secondary structure could be completely suppressed by the addition of guanidine hydrochloride to the sample (Fig. 2C).

The 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

Biological activity in vitro was tested by treating serum starved MCF7 cells with 5 μg/mL gAd in DMEM for 5 min. Western Blot analysis using phosho-ACC and ACC antibodies revealed that pH-solubilised gAd could significantly increase ACC phosphorylation about 1.5 fold over basal level (Fig. 3).

Recombinant gAd lowers blood glucose in STZ-induced type 1 diabetic mice

The biological activity of gAd as a blood glucose lowering agent in vivo was investigated in STZ-induced type 1 diabetic mice (Fig. 4). Animals showed a 50 % decrease in blood glucose levels (P=0.004) 4 h after treatment compared to unchanged levels in the saline treated control group (Table 1). Discussion

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.

In vivo and in vitro data has established adiponectin as a reliable biomarker for these disorders and an important element of metabolic improvement thus representing an intriguing 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.

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. For inclusion body derived adiponectin yields of 100 mg/L culture medium have been reported ⁹. Liu and Liu et al published the expression gAd in Pichia pastoris with yields of about 50 mg/L culture medium ⁵.

The present results provide access to much higher levels of the production of gAd. This is in agreement with data obtained for other proteins. Nine out of 31 therapeutic proteins approved from 2003 to 2006 are being produced in E. coli. The recovered gAd was of good purity without a chromatography step and biological activity was demonstrated in a cell culture model and in vivo. Recently high-cell-density E. coli expression protocols have been reported based on autoinduction or high-cell-density IPTG induction consistently yielding 280-680 mg/L cell culture for several proteins tested¹¹'¹². Recombinant gAd was expressed as inclusion bodies in E. coli B121(DE3)pLysSRARE as His-tag fusion protein using the pET15b expression vector.

The formation of 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. By applying 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. Importantly the homogenity in inclusion bodies is generally high reducing the need for subsequent purification steps.

To capitalise on the presence of extensive native-like secondary structures in inclusion body proteins, 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.

In the nineteen nineties several groups reported pH assisted solubilisation of inclusion bodies. While some alkaline pH assisted procedures still demanded high chaotropic agent concentrations (8 M urea) ¹³'¹⁴ a mild solubilisation strategy employing only 2 M urea or guanidine hydrochloride at pH > 12 has been applied for the recovery of human growth hormone (HGH) and zona pellucida protein ¹⁵'¹⁶. This mild solubilisation process without disturbing the native-like secondary structure significantly increased the recovery of bioactive HGH via pulsed renaturation process, simple dilution or dialysis. In contrast to our protocol all procedures required solubilisation times of at least 30 min to one hour increasing the possibility of irreversible modifications of the protein. Alkaline pH has also been patented to solubilise and refold proinsulin ¹¹.

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. Furthermore 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.

Taken together the present strategy already gives access to gram scale amount of active gAd out of 1 L expression culture without any optimization of expression time, temperature, culture medium etc. Using fed-batch fermentation could increase the yield to the magnitude of kilograms. This production scheme could clear the hurdle of limited access to active recombinant adiponectin by supplying sufficiently large amounts for the therapeutical application of this potent adipokine and promising pharmaceutical agent.

Table 1. Effect of gAd on blood glucose levels in STZ-induced type 1 diabetic mice

Blood glucose (mmol/1)

Oh 4h

Control (saline) 24.4 ± 4.5 25.5 ± 5.4

gAd 5mg/kg BW 21.7 ± 3.1 10.9 ± 3.7

References

1 Ouchi, N. et al. Adipocyte-derived plasma protein, adiponectin, suppresses lipid

accumulation and class A scavenger receptor expression in human monocyte-derived macrophages. Circulation 103, 1057-1063 (2001).

2 Fruebis, J. et al. Proteolytic cleavage product of 30-kDa adipocyte complement- related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98, 2005-2010 (2001).

3 Berg, A. H., Combs, T. P., Du, X., Brownlee, M. & Scherer, P. E. The adipocyte- secreted protein Acrp30 enhances hepatic insulin action. Nat Med 7, 947-953 (2001).

4 Yamauchi, T. et al. The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat. Med. 7, 941-946 (2001).

5 Liu, D. G. et al. Functional expression of the globular domain of human adiponectin in Pichia pastoris. Biochem. Biophys. Res. Commun. 363, 769-775 (2007).

6 Sulpice, T. et al. An Adiponectin-Like Molecule with Antidiabetic Properties.

Endocrinology 150, 4493-4501 (2009).

7 Arita, Y. et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 257, 79-83 (1999).

8 Wang, Y. et al. Post-translational modifications of the four conserved lysine residues within the collagenous domain of adiponectin are required for the formation of its high molecular weight oligomeric complex. J Biol Chem 281, 16391-16400 (2006).

9 Hu, X. B., Zhang, Y. J., Zhang, H. T., Yang, S. L. & Gong, Y. Cloning and expression of adiponectin and its globular domain, and measurement of the biological activity in vivo. Sheng Wu Hua Xue Yu Sheng Wu Wu LiXue Bao (Shanghai) 35, 1023-1028 (2003).

10 Tsao, T. S., Lodish, H. F. & Fruebis, J. ACRP30, a new hormone controlling fat and glucose metabolism. Eur J Pharmacol 440, 213-221 (2002).

11 Studier, F. W. Protein production by auto -induction in high density shaking cultures.

Protein Expr Purif 41, 207-234 (2005).

12 Sivashanmugam, A. et al. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18, 936-948 (2009).

13 Tichy, P. J., Kapralek, F. & Jecmen, P. Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies. Protein Expr Purif 4, 59-63 (1993).

14 Zhang, Z., Zhang, Y. & Yang, K. Mechanism of enhancement of prochymosin

renaturation by solubilization of inclusion bodies at alkaline pH. Sci China C Life Sci 40, 169-175 (1997).

15 Patra, A. K., Gahlay, G. K., Reddy, B. V., Gupta, S. K. & Panda, A. K. Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli. Eur J Biochem 267, 7075-7081 (2000).

16 Sonoda, H. & Sugimura, A. Improved solubilization of recombinant human growth hormone inclusion body produced in Escherichia coli. Biosci Biotechnol Biochem 72, 2675-2680 (2008).

17 Hartman, J., Mendelovitz, S. & Gorecki, M. Refolding of proinsulins without addition of reducing agents. United States patent 6001604 (1999).

Claims

P28761 Patent Claims:

1. A method of producing a soluble recombinant protein by in vitro folding, comprising the steps of:

2. The method of claim 1 , wherein the recombinant protein is selected from human proteins, fusion proteins and fragments thereof.

3. The method of claim 1 or 2, wherein the recombinant protein is selected from adiponectin or adiponectin-like protein.

4. The method of claim 3, wherein the adiponectin-like protein is selected from the group consisting of Ci /77VF-related proteins.

5. The method of claim 4, wherein the adiponectin-like protein is selected from Clq and tumor necrosis factor related protein 1-9.

6. The method of one or more of the preceding claims, wherein the bacterial host is E. coli or any other enter ob acteriaceae which is capable to produce proteins in insoluble form.

7. The method of one or more of the preceding claims, wherein adding the alkaline agent in step b) adjusts the pH of the solution to 12-13, preferably 12,5.

8. The method of claim 7, wherein the alkaline agent is selected from NaOH, KOH or NH₄OH.

9. The method of claim 7 or 8, wherein the solvent is a buffer solution, preferably TRIS, hepes or phosphate buffer.

10. The method of one or more of the preceding claims, wherein the solution obtained in step b) is centrifuged, the supernatant is removed and filtered and further used in step c).

11. The method of one or more of the preceding claims, wherein the organic solvent added in step c) is acetone.

12. The method of one or more of the preceding claims, wherein no chaotrope agents are used.

13. A recombinant protein, obtainable by the method of one or more of claims 1-12.

14. A pharmaceutical composition, comprising the recombinant protein as defined in claim 3 and a suitable pharmaceutically acceptable carrier or diluent.

15. The pharmaceutical composition of claim 14 for use in the treatment of obesity-related metabolic disorders.