Refolding and purification of recombinant human granulocyte colony-stimulating factor using hydrophobic interaction chromatography at a large scale

High level expression of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in Escherichia coli (E. coli) usually forms insoluble and inactive aggregates, i.e. inclusion bodies. In the present work, high performance hydrophobic interaction chromatography (HPHIC) was applied to the refolding of rhG-CSF, which was solubilized by 8.0 mol L^?1 urea from the inclusion bodies. First a laboratorial scale column (10 mm × 20 mm I.D.) was employed to study the refolding process. Several factors, including concentration of ammonium sulfate, pH of the mobile phase and flow rate, were investigated in details. The results indicated that the rhG-CSF produced by E. coli could be successfully refolded with simultaneous purification by using HPHIC. The refolding process was further scaled up by using a large column (50 mm × 200 mm I.D.). 200 mL of rhG-CSF solution solubilized by 8.0 mol L^?1 urea, with a total amount of protein around 1.6 g, could be loaded onto the large column at one time. Under these conditions, the obtained rhG-CSF had a specific activity of 2.3 × 10⁸ IU mg^?1 and a purity of 95.4%, the mass recovery during the purification was 36.9%. This work might have great impact on practical production of rhG-CSF, and it also shed a light on protein refolding using liquid chromatography at large scales.     

Expression of recombinant human cathepsin K is enhanced by codon optimization

A synthetic codon-optimized gene encoding human procathepsin K has been cloned in Escherichia coli using pET28a+ vector. The recombinant His-tagged fusion protein was expressed as inclusion body, solubilized in urea and purified by metal affinity chromatography. The purified protein was refolded by dilution technique, concentrated and finally purified by gel-filtration chromatography. The expressed protein was confirmed by Western blot analysis with human cathepsin K specific antibody. We have obtained 140 mg purified and refolded protein from 1 L bacterial culture which is the highest (nearly three times higher) yield reported so far for a recombinant human procathepsin K. The protease could be autocatalytically activated to mature protease at lower pH in presence of cysteine protease specific activators. The recombinant protease showed gelatinolytic and collagenolytic activities as well as activity against synthetic substrate Z-FR-AMC with a K_m value of 5 ± 2.7 μM and the proteolytic activity of the enzyme could be blocked by cysteine protease inhibitors E-64, leupeptin and MMTS.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro.     

Purification and efficient refolding process for recombinant tissue-type plasminogen activator derivative (reteplase) using glycerol and Tranexamic acid

In this study, a novel and economic method for refolding and purifying recombinant tissue plasminogen activator derivative (r-PA; reteplase) was developed. Reteplase with nine disulfide bonds in its complex structure is expressed in the form of inclusion bodies in Escherichia coli and requires tedious dissolving and refolding processes to achieve its biological activity. Among the different refolding additives that were evaluated, glycerol and tranexamic acid (Txa) were found to be more effective in increasing the refolding yield of reteplase. Using response surface methodology, a solution containing 3.5 M urea, 33% (v/v) glycerol, and 400 mM Txa was found to give the highest refolding yield. The synergic effect of urea, glycerol, and Txa under optimum conditions for a reteplase concentration of 25 μg ml⁻¹ resulted in a high refolding yield of 76.41%. Increased reteplase concentration in the refolding buffer was achieved using the pulse-fed method. In the pulse-fed method, a refolding yield of 49.53% was achieved for a final reteplase concentration of 300 μg ml⁻¹. Using Txa as a novel refolding aid for reteplase instead of ionic amino acids like l-Arginine allowed to purify the refolded reteplase directly by cation-exchange chromatography with high purity.     

Membrane combined with hydrophilic macromolecules enhances protein refolding at high concentration

Membrane technology is important to the development of modern biotechnology. It has the potential to efficiently refold protein at high concentration that is still a challenge for pharmaceutical protein produced from inclusion bodies. This paper dealt with the application of a polysulfone hollow fiber membrane to protein refolding using recombinant human granulocyte colony-stimulating factor (rhG-CSF) as a model protein. Compared with dilution refolding at protein concentration of 1.0 mg/mL, the crossflow membrane system led to a 16% increase in soluble protein recovery, and a 3.3-fold increase in specific bioactivity. Addition of PEG 6 K at 2 g/L could further improve the soluble protein recovery up to 57%, the specific bioactivity up to 2.2 × 10⁸ IU/mL. Addition of dextran at 5 g/L could increase the soluble protein recovery up to 63.6%, the specific bioactivity up to 2.30 × 10⁸ IU/mL. By gently and gradually removing denaturant, ultrafiltration membrane system was demonstrated to be very helpful for protein refolding at high concentration. Combining with hydrophilic macromolecular of PEG or dextran could further increase its efficiency. PEG was able to promote the refolding intermediate of rhG-CSF to transfer into the native structure; whereas dextran could enhance protein refolding mainly by weakening shear stress-induced protein aggregation.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

Enhanced extraction genistein from pigeon pea [Cajanus cajan (L.) Millsp.] roots with the biotransformation of immobilized edible Aspergillus oryzae and Monacus anka and antioxidant activity evaluation

A new method of enhanced extraction genistein from pigeon pea [Cajanus cajan (L.) Millsp.] roots with the biotransformation of immobilized edible Aspergillus oryzae and Monacus anka, was investigated. It showed that immobilized Aspergillus oryzae and Monacus anka on sodium alginate effectively supported the highest genistein extraction yield by screening microorganism tests. After biotransformation process with immobilized Aspergillus oryzae and Monacus anka under 30 °C, pH 6.0, 2 days, liquid-solid ratio 12: 1 (mL/g), the extraction yield of genistein reached 1.877 mg/g, which was 2.65-fold to that of normal extraction yield. Moreover, IC₅₀ values of the extracts measured by DPPH-radical scavenging test and β-Carotene-linoleic acid bleaching test were 0.737 mg/mL and 0.173 mg/mL (control sample 1.117 mg/mL and 0.216 mg/mL), respectively. SOD (Super Oxygen Dehydrogenises) activity of the extracts treated with immobilized microorganism which was stronger than that of the untreated pigon pea roots (1.44 U/mg) at the concentration of protein (0.9375 μg/mL) was 1.83 U/mg. The developed method could be an alternative method for the enhanced extraction of genistein from plants and could be potentially applied in the food industry     

Enzymatic synthesis of cyclohexyl-α and β-d-glucosides catalyzed by α- and β-glucosidase in a biphase system

Two secondary alcohol glucosides, cyclohexyl-α-d-glucoside and cyclohexyl-β-d-glucoside, were synthesized via the condensation reaction of cyclohexanol with d-glucose in a biphase system catalyzed by α-glucosidase and β-glucosidase, respectively. The effects of pH, water content, glucose concentration and metal ions on the yield of glucosides were studied. The optimum catalytic conditions established for α-glucosidase was 25% (v/v) water content, 2.5 mol/L glucose concentration and pH 2.0, and for β-glucosidase was 30% (v/v) water content, 2.0 mol/L glucose and pH 5.0. The maximum yield of glucoside was 13.3 mg/mL for cyclohexyl-α-d-glucoside and 8.9 mg/mL for cyclohexyl-β-d-glucoside. Synthesis progress was monitored by TLC and quantitatively analyzed by pre-derived capillary gas chromatography (GC). The retention time was 12.34 min for the α isomer and 12.96 min for the β isomer, respectively. With an anomeric purity of more than 99.5%, the two glucosides display excellent site-specific catalysis by α- and β-glucosidase. Herein, we present a general method to produce anomerically pure glucosides via a one-step bio-reaction in a biphase system. This method could potentially be applied in glucosylation of primary and secondary alcohols or other reactions requiring glucosylation.     

An analysis of the factors that affect the dissociation of inclusion bodies and the refolding of endostatin under high pressure

An optimization of the refolding of endostatin (ES), by a study of the conditions that can affect (i) dissociation of inclusion bodies (IBs) and (ii) renaturation under high hydrostatic pressure (HHP), is described. IBs produced by bacteria cultivated at 25 °C were shown to be more soluble than those produced at 37 °C and their dissociation by application of 2.4 kbar at 20 °C was shown to be further enhanced at ?9 °C. A red shift in intrinsic fluorescence spectra and an increase in binding of the hydrophobic fluorescent probe bis-ANS show subtle changes in conformation of ES in the presence of 1.5 M GdnHCl at 2.4 kbar, while at 0.4 kbar the native conformational state is favored. The 25% refolding yield obtained via compression of IBs produced at 37 °C by application of 2.4 kbar, was increased to 78% when conditions based on the insights acquired were utilized: dissociation at 2.4 kbar and ?9 °C of the IBs produced at 25 °C, followed by refolding at 0.4 kbar and 20 °C. Besides providing insights into the conformational transitions of ES structure under HHP, this work proposes innovative conditions that are likely to have wide applicability to the HHP-induced refolding of proteins in general.     

Refolding of recombinant human interferon gamma inclusion bodies in vitro assisted by colloidal thermo-sensitive poly(N-isopropylacrylamide) brushes grafted onto the surface of uniform polystyrene cores

Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γ in vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55 nm and 110 nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33 °C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50 μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15 °C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.     

Soluble expression and purification of a functional harpin protein in Escherichia coli

The use of harpins in practical agricultural applications may enhance plant growth and induce disease resistance. However, few investigations focused on the optimal expression and purification of harpin. In this work, harpin protein fused with a thioredoxin tag and a hexahistidine tag was expressed in Escherichia coli BL21 (DE3) cells as a soluble form under the induction of 0.5 mmol/L isopropyl β-D-1-thiogalactopyranoside. The purity of the recombinant harpin was greater than 90% after one-step nickel-nitrilotriacetic acid affinity chromatography. The yield of purified TRX-harpin protein reached 17.1 mg per 100 mL of cell culture. TRX-harpin is thermostable and could trigger the hypersensitive response effect in tobacco, with an efficient dose as low as 30 μg/mL. The root lengths of TRX-harpin treated tobacco and wheat plants were nearly 1.6-fold and 1.8-fold longer, respectively, than plants treated with the empty vector preparation. Thus, using a N-terminal TRX-tagged fusion is an economic way to produce bioactive harpin.     

High productivity refolding of an inclusion body protein using pulsed-fed size exclusion chromatography

Protein refolding using size exclusion chromatography (SEC) is advantageous over conventional refolding methods in terms of ease of automation, simultaneous purification capabilities, and the non-adsorptive protein–matrix interaction which eliminates steric constraints. Despite these advantages, the widespread use of SEC refolding remains restricted by low process productivity and product concentration bottlenecks. This study aims to address those limitations and exploit SEC advantages for large-scale refolding applications. Specifically, this study reports the development of a pulsed-fed size exclusion chromatography (PF-SEC) refolding platform, which successfully refolded E. coli-derived α-fetoprotein (AFP) to achieve 53% refolding yield at 0.9 mg/ml AFP refolding concentration. AFP was introduced into the column by pulsed injection to increase feed load, while suppressing concentration-induced aggregation. Refolding was initiated by a urea gradient in the column, where the gradient length could be readily adjusted to complement pulsed feeding patterns. AFP refolding productivity with PF-SEC improved by 8- and 64-fold compared to ion-exchange chromatography refolding and pulsed dilution refolding, respectively, at a fixed refolding concentration. Through a unique integration of pulsed feeding and urea gradient development, this new PF-SEC refolding methodology overcomes ‘productivity and concentration’-related disadvantages inherent in SEC, and will be scalable for large-scale protein refolding applications.     

The study on microwave assisted enzymatic digestion of ginkgo protein

Microwave-assisted enzymatic digestion (MAED) technique was applied for ginkgo protein digestion with both free and immobilized enzyme. Under the optimized conditions of MAED (0.01 g/mL substrate concentration of bromelain, 4500 U/g enzyme/ginkgo protein, 30 min, 300 W microwave power), a higher digestion rate (7.50%) and a significant increase in antioxidant activity (72.7 mg/g) were obtained in contrast with the conventional methods. With the optimized digestion conditions (0.625% glutaraldehyde (v/v), 0.4 mg/mL initial concentration of bromelain and 4 h of immobilization), the activity and effectiveness factor of immobilized bromelain were respectively 86 U and 81.6%. The results of ginkgo digestion by applying MAED indicated that the digestion rate of immobilized bromelain obtained by MAED method (6.41%) was comparable to that of free bromelain in the conventional digestion (8.13%). In both case with immobilized and free bromelain while applying MAED, a homogeneously abundant distribution of peptide fragments (from 7.863 Da to 5856 Da) and a few different peptide profiles were found. This report brings in conclusion that applying MAED with immobilized enzyme has the potential to obtain the highest number of antioxidant activity peptides.     

Effect of chaotropes in reverse micellar extraction of kallikrein

Reverse micellar extraction is a promising technique in large-scale bioseparation. However, low recovery and high salt concentration in back extraction limit its application. In CTAB/n-octane/n-hexanol reverse micellar system, the enzyme, pancreatic kallikrein could be effectively enwrapped into reverse micelles in forward extraction, but was difficult to be released during back extraction. In this study, dilute chaotropes (urea and GuHCl) were introduced to enhance the release of enzyme instead of high salts in back extraction. Kallikrein enwrapped in reverse micelles was released effectively in the presence of dilute urea and GuHCl during back extraction. Nearly 100% activity recovery of kallikrein from commercial product was obtained by adding 0.60 M urea, and for kallikrein from crude material, the recovery increased greatly by adding 0.80 M urea and 0.08 M GuHCl in the stripping solution. The mechanism of chaotrope for enhancing the release of enzyme from micelles was explored and dynamic light scatter analysis showed that the chaotrope would influence the sizes of micelles during reverse micellar extraction.     

Fast biodegradation of long chain n-alkanes and crude oil at high concentrations with Rhodococcus sp. Moj-3449

Biodegradation of long chain n-alkanes and crude oil with fast rate and high concentration are desirable for bioremediation, especially in heavily oil-polluted areas, and enhanced oil recovery. We discovered Rhodococcus sp. Moj-3449 with such unique abilities by screening microorganisms for the growth on n-hexadecane at 30 mg/mL. The new strain grew very fast on 120 mg/mL of n-hexadecane giving a cell density of 14.7 g cdw/L after only 2 days’ incubation. During the growth with this strain, the oil–water phases were rapidly emulsified, giving rise to tolerance to high alkane concentration (250 mg/mL) and fast growth rate of 0.10–0.20 h^?1 for alkane concentration of 1–180 mg/mL. The degraded concentration of n-hexadecane increased linearly with the initial alkane concentration (1–250 mg/mL). Incubation on n-hexadecane at 250 mg/mL for 7 days gave a cell density of 13.5 g cdw/L and degraded 124 mg/mL of n-hexadecane. The strain grew also fast on n-dodecane (C12), n-tetradecane (C14), and n-octadecane (C18), with degradation preference of C14 (=C16) > C12 > C18. Different from many alkane-degrading strains, Rhodococcus sp. Moj-3449 was found to have subterminal oxidation pathway. Rhodococcus sp. Moj-3449 degraded also crude oil fast at 60–250 mg/mL, with a wide range of n-alkanes (C10–C35) as substrates in which C14–C19 are preferred. The degradation ability increased with initial oil concentration from 60 to 150 mg/mL and slightly decreased afterwards. Incubation on 150 mg/mL of crude oil for 7 days degraded 37% of n-alkanes. The outstanding ability of rapidly degrading long chain n-alkanes and crude oil at high concentration makes Rhodococcus sp. Moj-3449 potentially useful for bioremediation and microbial enhanced oil recovery.     

Expression,refolding and purification of a human interleukin-17A variant

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T₇ promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris–HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2–8 °C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.     

Chemical composition,angiotensin I-converting enzyme (ACE) inhibitory,antioxidant and antimicrobial activities of the essential oil from south Tunisian Ajuga pseudoiva Rob. Lamiaceae

The essential oil of Ajuga pseudoiva, collected from Tunisia, was analyzed using gas chromatography–mass spectroscopy. Thirty-two compounds accounting for 95.76% of the total oil were identified. Sesquiterpenes were found to be the most abundant components of A. pseudoiva oil. And they were mainly represented by viridiflorol (30.17%), germacrene B (9.26%) (α, β and γ)-eudesmol (8.11%) and aromadendrene (7.45%). The essential oil of A. pseudoiva showed radical scavengers activity (IC₅₀ = 0.72 mg/mL) and displayed lipid peroxidation inhibitory activity (IC₅₀ = 0.6 mg/mL). A. pseudoiva essential oil was also found to exhibit a dose-dependent ACE inhibitory activity with an IC₅₀ value of 65.5 μg/mL. Moreover, the antimicrobial activity of the essential oil was tested against 17 species of microorganisms, and the results obtained showed significant antibacterial activity against the Gram-positive and Gram-negative bacteria, with inhibition zones and minimal inhibitory concentration values of 14–32 mm and 84–137 μg/mL and 6–21 mm and 105–336 μg/mL, respectively. Higher activity was also found against several fungal strains.     

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO₄ and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.     

Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry

A sensitive, specific and selective method has been developed for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma. The method employed a state of the art LC–MS/MS operated in the positive and negative ionization switching modes. A simple sample preparation step involving protein precipitation with acetonitrile has been optimized; the analytes and the internal standard moxifloxacin were separated on a Purosphere^® STAR C₈ column (125 mm × 4 mm, 5 μm). The mobile phase was an ammonium acetate solution (1 mM) with formic acid (0.2%): methanol and acetonitrile (65:17.5:17.5, v/v/v (%)), the flow rate was set at 0.65 mL/min. Bisoprolol and hydrochlorothiazide were ionized using ESI source prior to detection by Multiple Reaction Monitoring (MRM) mode while monitoring at the following transitions: positive m/z 326 → 116 for bisoprolol, negative m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide. Linearity was demonstrated over the concentration range 0.10–30.0 (ng/mL) for bisoprolol and 1.00–80.00 ng/mL for hydrochlorothiazide. The limits of detection were 0.100 (ng/mL) for bisoprolol and 1.00 (ng/mL) for hydrochlorothiazide. The validated method was successfully applied to a pharmacokinetic study of 5 mg bisoprolol fumarate with 12.5 mg hydrochlorothiazide tablet in healthy volunteers.     

Cost effective characterization process and molecular dynamic simulation of detergent compatible alkaline protease from Bacillus pumilus strain MP27

A psychrothermotolerant alkaline protease isolated from Bacillus pumilus MP27 with a molecular mass ∼53 kDa was isolated from Southern ocean water samples. It was partially purified by single step TPP with purity fold of 16.65. The enzyme was found to be widely stable within a range of temperature and pH, maintaining 52.25% of its activity at 50 °C and 92% at pH 12. The enzyme exhibited an exceptional activity along with tested detergents, showing 98% stability with SDS (10 mg/ml) and ̴ 99% stability with Tide detergent (7 mg/ml). Further, the alkaline protease gene of 1152 bp was successfully cloned in pGEM-T Easy vector in E. coli DH5α. The gene sequence was further translated, modeled and molecular dynamic simulation was performed. The modeled protein was highly unstable during the first 5 ns and therefore could not able to form bonds with the ligand after 1 ns of simulation.