| Protein-based therapeutics is emerging as the largest class of new chemical entities being developed and researched. Transferring and expressing desired genes in a foreign cell can be efficiently achieved by using the technology of recombinant DNA. However, the host cell greatly affects the quality and quantity of the produced recombinant protein. The productions of recombinant mammalian-derived proteins in bacteria, e.g. Escherichia coli, do not always product a biologically active, soluble protein because of the different mechanisms of protein translation, modification and folding between prokaryotic cell and eukaryotic cell.Recombinant tissue plasminogen activator variant (Reteplase) represent one of the third generation thrombolytic drugs. Reteplase manifests the advantages in both manufactary and clinic: large scale production, prolonged half-life time, thrombolytic potency and fewer side effects. Therefore, it attracts great attention from cardiovascular experts. Reteplase is the non-glycosylated, truncated mutant of wild-type t-PA molecule. It is a domain-delete protein variant, which only contains kringle-2, protease domain and N-terminus three amino acids. The protein sequence contained 355 amino acids, 18 cysteines and 9 disulfide bonds with a calculated MW of 39,000 Daltons. In vitro, refolding yield of Reteplase is very low.Although a variety of hosts may be used to produce these proteins, E.coli is still the first choice, particularly when the biological activity of the protein product is not dependent on post-translation modification. Recombinant proteins can be expressed at high concentration levels. In many cases, in E.coli, the accumulation of the protein product in inactive insoluble deposits inside the cells, called inclusion bodies, often occurs. A variety of means have been tried by researchers to improve solubility and activity, but the optimal process for a specific product is still unpredictable. We have tried various expressing systems, including eukaryotic system Pichia pastoris, E.coli host with plasmid containing signal peptide. However, the results are not satisfied. The purification and the refolding of IB proteins is an attractive alternative because that the aggregates can be easily separated and mostly contain the product with a high concentration. The in vitro refolding process is the critical step, but the optimisation can be performed in a strategic way with a. step by step evaluation of the optimum conditions and additives contrarily to the in vivo strategy. If effective technique of protein refolding has been developed, satisfied products are expected using inclusion body. This is the main reason for using E.coli BL21 and pET22b plasmid containing T7 promoter to produce bioactive proteins in vitro.Inclusion bodies have been used extensively as a source of relatively pure, misfolded polypeptide chains that can be renatured to give the biologically active soluble protein. Expressing a protein in the format of inclusion body has its obvious advantages. Large amounts of highly enriched proteins can be expressed as inclusion bodies. Trapped in insoluble aggregates, these proteins are for the most part protected from proteolytic degradation. If the protein of interest is toxic or lethal to the host cell, then inclusion body expression may be the best available production method. Furthermore, the optimisation of the inclusion body expression conditions is sample as the parameters are limited, in contrast to the complexity of optimising soluble in vivo production. The fermentation process of recombinant Reteplase expression in E.coli BL21 was studied. The effects of influencing factors, including concentration of IPTG, induction time, cultivation temperature and feeding strategies, have been explored. The results suggest that an OD of 40 for 2L fermentator and expressing rate of around 30% was achieved, with the condition of IPTG 80μM, cultivation time after induction between 4～5 h, glucose concentration under 0.1 g/L, 40℃, oxygen dissolve remaining 20%. Using sample protocol, we have been able to obtain 80% purification inclusion bodies, which were prepared for refolding. The recovery of bioactive proteins from inclusion bodies is a complex process that should be carefully manipulated. The challenge is to take advantage of the high expression levels of inclusion body proteins by being able to convert inclusion body into soluble bioactive proteins.In order to explore the efficient methods of in vitro protein refolding, three methods in refolding Reteplase in vitro were chosen to do a comparison. First method is a traditional dilution refolding. Direct dilution the oxido-shuffling reagents GSH/GSSG was utilized to promote both formation and reshuffling of disulfide bonds. L-Arginine was added to refolding buffer to prevent aggregation. Second one is protein disulfide isomerase (PDI) catalyzes protein refolding. Third one is protein refolding on column. Revised size exclusion chromatography (SEC) was used to assist Reteplase refolding.The simplest refolding procedure is to dilute the concentrated protein-denaturant solution into a refolding buffer that allows the formation of the native structure of the protein. This technique has serious drawbacks during scale-up as huge refolding vessels and additional cost-intensive concentration steps are required after refolding. Different protein concentrations, different ratios and concentrations of GSH/GSSG, concentrations of L-Arginine, and refolding time scale have been optimized in this study. When Reteplase protein concentration was 0.1 mg/ml, average refolding yield reached about 2%.PDI is an enzyme that catalyzes disulfide formation and isomerization and also a chaperone that inhibits aggregation. When the molar concentration ratio between rhPDI and Reteplase was 1:50, average refolding yield reached about 13.2%. Because chaperones and foldases are proteins that need to be removed from the refolding solution at the end of the refolding process and as they may be costly to produce, their commercial use will require a recovery-reuse scheme. The application of chaperonin systems on an industrial scale is limited because they act in a stoichiometric manner thus producing high costs.Refolding using packed columns is attractive because it is easily automated using commercially available preparative chromatography systems. There are a few advantages of refolding proteins on columns: 1) scale invariant - results from screening can be translated to manufacture without significant changes in the technology used; 2) easily automated - to address the issue of speed and to enable high-throughput processing of samples; 3) generic for a broad range of similar proteins; After compare principally different approaches of chromato- graphic refolding, size exclusion chromatography (SEC) was selected to refold Reteplase.A revised SEC on Sephadex G25-M was used to refold Reteplase. In order to raise the refolding rate, a Superdex G25-M column was pre-equilibrated with refolding buffer, a linear decreased GuHCl gradient, a linear increased PH gradient and a dual-gradient of decreasing refolding concentration, combined with an increasing pH-gradient separately. By controlling the loading of the protein and flow-rate, the refolding process can be carefully kinetically controlled. The comparison of the different refolding processes shows the GuHCl gradient gel filtration and a dual-gradient gel filtration have the best result with regard to activity recovery. When the sample is eluted, the gradient moves downwards as well. However, the protein moves faster than the low molecular weight reagents gradient. At the end of the column, the protein has passed through the gradient, refolded, and left the column. By utilizing SEC assistanting Reteplase refolding, we can obtain yield of refolding 9.2%(protein concentration as 0.32mg/ml). As compared with retplase produced by Boehringer Mannheim, which refolding yield is 5%, our system have higher both refolding yield and final protein concentration.The ETI-Sepharose4B ETI affinity chromatography column has been used to purify Reteplase. ETI is a kind of serine proteinase inhibitor, which has the ability to inhibit Reteplase. ETI protein was obtained by genetically engineered technology in P. pastoris then conjugated to CNBr-Sepharose4B to make an affinity chromatography column. The conclusion can be easily drawn that, among the three methods of refolding Reteplase, refolding using packed columns has obvious advantages in various ways, including high refolding rate, less complicated technique, and more cost-effectiveness. This study has its significance both in theoretical research and practical application.In the end, a summary of the study has been given. Meanwhile, the directions of future studies have been prospected based on the review of recent progress of protein refolding research.
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