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Studies Related to Common Issues and Specific Issues in the Human Serum Albumin Fusion Platform

Author: WuMin
Tutor: ChenShuQing
School: Zhejiang University
Course: Pharmacology
Keywords: human serum albumin fusion protein linker ELISA Pichia pastoris efficient expression protease copy number chaperone
CLC: Q51
Type: PhD thesis
Year: 2013
Downloads: 39
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Abstract


1. The optimal design of the long-acting HSA/GH fusion proteinTherapeutically relevant proteins and peptides are playing an increasing role in clinical use. The development of therapeutic peptides or proteins, however, is often impeded by their short half-lives and frequent administration, e.g. hGH (T1/2<20min in human body) and PTH (1-34)(T1/2≈5min by intravenous administration). Human serum albumin fusion technology is a simple and flexible method to extend circulatory half-lives of such proteins and peptides. Though attractive, there still remain some basic issues to be solved in the albumin fusion platform, e.g. the active sites of target proteins or peptides have been reportedly interfered by the huge HSA molecule, resulting loss of biological activities in vitro to a varying degree. In some worse cases, the correct folding of the target protein was partially affected resulting heterogeneity of the recombinant fusion protein. Thus, different fusion orientation and linker selection should be considered in the design of HSA fusion proteins. In this study, in order to obtain an optimized form of long-acting GH analog using HSA fusion technology, flexible linkers with different lengths were inserted between HSA and GH. The stabilities and the biological activities in vitro were compared among the designed fusion proteins, as evaluation indexes for the preferred fusion design. Besides, Data obtained with HSA and GH fusion design may also be helpful to other HSA fusion proteins.2. Establishment of immunoassay for HSA/GH fusion proteinA novel sandwich ELISA was established and optimized to determined the fusion protein of HSA/GH and the detection range was2.156~69ng/ml. This method has been successfully applied to pharmacokinetics study in rats and could aid the preclinical study of HSA/GH fusion protein.3. Establishment of high-performance Pichia pasrotis system for recombinant expression of HSA fusion proteinsYeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secret and modify foreign proteins according to a general eukaryotic scheme. The fact that Pichia pastoris has been considered as the most efficient and economical expression system for the production of HSA, indicates the potential advantages for expression of HSA fusion proteins using Pichia pastoris. In our study, Pichia pastoris has been used to express a series of HSA fusion proteins. However, two major problems were encountered when using the un-optimized Pichia pastoris, which seriously hampered the preclinical process of HSA fusion proteins. The first problem is proteolysis of secreted HSA fusion proteins and the second one is inefficient secretion of HSA fusion proteins. Thus, it is necessary to optimize Pichia pastoris system to achieve high-level expression of HSA fusion proteins.3.1Reducing the proteolytic degradation of HSA fusion protein by construction of protease-deficient strainThe degradation appears to be more severe when the fusion partner is a proteases-sensitive protein or peptide. In this study, HSA/PTH (1-34) was chosen for the detailed proteolysis study since it suffered the most severe degradation.Firstly, the secreted products (both intact protein and degraded proteins) were characterized by Western Blot, N-terminal protein sequence and MS analysis. Secondly, the suspicious proteases were studied by construction of corresponding protease-deficient strain or using the commercialized strain. These proteases include yeast yapsins (YPS1, YPS2, YPS3, YPS7, MKC7, YPS’,YPS") and two major vacuolar proteases (PrA and PrB). As a result, we have successfully identified that both PrA disruption and YPS1disruption were beneficial for degradation reduction of HSA/PTH (1-34) fusion protein. Degradation reduced to minimum when simultaneously disruption of PrA and YPS1, with the recovery of intact protein increased from30%to80%, as compared with the wild-type GS115.3.2Expression improvement of HSA fusion proteins by combination of multi-copy HSA fusion genes integration and co-expression with chaperones.In the preliminary study, a series of HSA fusion proteins have been successfully secreted from wild-type strain or protease-deficient strain, including HSA/GH, HSA/PTH (1-34), HSA/ILIRa and HSA/Thymosin αl. However, the secretion level is generally low (25-50mg/L), which limits the further fermentation optimization. The low expression level of HSA fusion proteins is a drag on the process of preclinical studies, which also increase the cost of follow-up industrial production. Therefore, construction of high-level expression strains is particularly important.In this study, the effect of copy number of HSA fusion gene was evaluated first. The results proved the higher expression with increased HSA fusion gene copy number. Co-expression with several important chaperones based on the strain containing multi-copy HSA fusion genes would further improve the expression level. This method could be applied to a series of HSA fusion proteins.

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