Effects of b-cyclodextrin, bCD, on Refolding of lysozyme was investigated at pH 12 employing isothermal titration calorimetry (ITC) at 300K in 30mM Tris buffer solution. bCD was employed as an anti-aggregation agent and the heats obtained for lysozyme+bCD interactions are reported and analyzed in terms of the extended solvation model. It was indicated that there are two sets of identical and non-cooperative sites for bCD. Enthalpic force in the first binding sites is more important than entropic one, indicating that electrostatic interaction plays an important role in the interaction of lysozyme with bCD. The interaction in the second binding sites is stronger and both enthalpy and entropy driven but hydrophobic interaction has more important than electrostatic force. These results suggest that the effects of bCD on lysozyme Refolding are attributed to its ability to suppress aggregation of the protein.

Low molecular size additives such as L-arginine and the redox compounds have been used both in the culture medium and in vitro Refolding to increase recombinant proteins production. Additives increase protein Refolding and yield of active proteins by suppressing aggregate formation or enhancing Refolding process.In this work, a comparative study was performed on Refolding of recombinant plasminogen activator (rPA) in the presence of different concentrations of denaturants and additives. Escherichia coli-expressed rPA inclusion bodies were solubilized in chaotropic denaturants and subjected to protein Refolding by dilution method. The effects of various additives, the impact of pH, residual Guanidin Hydrochloride (Gn-HCl) and Dithiothreitol (DTT) on Refolding process were investigated.The Refolding process was assessed by determination of protein solubility and biological assay. The results of the study demonstrated that the best condition for solubilizing the rPA inclusion body was 6M guanidine hydrochloride at pH=10. In Refolding step, Larginine showed increasing effect on suppression of aggregation at concentrations of 200-1000 mM.Glutathione pairs (GSH-GssG) showed Refolding enhancer effect in a range of 2-20 mM. The highest Refolding yield was obtained in 500 mM L-arginine and reduced/oxidized glutathione 10: 1 ratio in pH 10. In conclusion, the results show that L-arginine plays an important role in the Refolding of human PA, preventing the aggregation of folding intermediate, and glutathione pair is essential for the correct Refolding. The results also revealed that higher solubility in the presence of higher concentration of L-arginine (>500 mM) or pH (>10) is not associated with higher activity.

Background: Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra-molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed inE. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins.Methods: Dilution method that allows Refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to Refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously.Results: After protein solubilization and Refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots.Conclusion: By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins.

Background: Vascular endothelial growth factor (VEGF) plays an important role in development of new blood vessel and angiogenesis. Human VEGF121 is smallest member of VEGF family. Production of active and correct form of VEGF is the most challenging issue. Methods: Here we described a method for optimization of Refolding of VEGF121 which was expressed in bacterial host. Enzyme linked immunosorbent assay and proliferation assay of human endothelial cells was performed to monitor Refolding and functional assay of VEGF121. Results: Using described method; VEGF was in correct fold and detected by antibody in ELISA. Furthermore, VEGF stimulated proliferation of human endothelial cells in dose-dependent manner. Conclusion: Refolded VEGF has potential for stem cell differentiation.

Background: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and Refolding process limits production and purification of recombinant BMP-2 in bacterial systems.Methods: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in Origami TM E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and Refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line.Results: No difference was observed between oxidoreductase systems in improving the efficiency of protein Refolding. However, comparisons between two Refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein.Conclusion: A new method for production of biologically active dimeric form of BMP-2 inE. coli expression system was established in this study.

Background: Interferon α-2b is a vital biotherapeutic produced through the recombinant DNA technology in E. coli. The recombinant IFN-α 2b normally appears as intercellular IBs, which requires intensive Refolding and purification steps. Method: Purification of IFN-α 2b from solubilized IB was performed using two-phase extraction. To optimize Refolding conditions, the effects of pH and different additives, including cysteine, cystine, urea, glycerol, Triton X-100, NaCl, and arginine, were investigated. Optimal Refolding buffer (0. 64 mM of urea, 5. 57 mM of cysteine, and 1. 8 mM of cystine) was obtained using RSM. The Refolding process was performed by an optimized Refolding buffer in the dilution and fed-batch Refolding method at different protein concentrations (251000 µ g/mL). Result: At a final protein concentration of 500 µ g/mL, the fed-batch Refolding method yielded in a biological activity of 2. 24 × 10 8 IU/mg, which was nearly twice that of dilution method. Conclusion: Fed-batch Refolding method resulted in the biologically active IFN-α 2b with high purity, which can be used for research and industrial purposes.

Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and Refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for Refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the Refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for Refolding of solubilized reteplase was found to be 1. 15 M glucose, 9. 16 mM imidazole, and 0. 16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and Refolding.