High level production of recombinant healthcare proteins in Escherichia coli

Abstract

Functional healthcare proteins, provide functional benefit to human, is essential bio-material in future bio-industry. They consist of enzymes, antibodies, growth factors, and cytokines to influence the cellular process and immune response and can be used in cosmeceutical, biopharmaceutical, biomarkers, and protein therapeutics. The eukaryotic cells were used to produce functional healthcare proteins due to the ability of the post-translational modification (PTM). However, because the production yield difficult to satisfy the demand, the prokaryotic expression system is still used as an attractive tool. For the effective production of functional healthcare proteins in Escherichia coli (E. coli) without loss of function, the protein tag and/or chaperone was used to increase solubility, purification efficiency, and production yield. Therefore, I designed fusion protein to produce target protein in the cytoplasm of E. coli. In chapter 2, commonly used proteases, tobacco etch virus (TEV) protease and human enterokinase light chain (hEK$_L$) were produced in E. coli. I focus on the production of hEK$_L$ in E. coli which is needed for novel strategies which are fusion with maltose-binding protein, replacement of cysteine with serine, using the SHuffle as a host cell, and co-expression of chaperone. In chapter 3, I designed the fusion protein to produce the recombinant N-terminal pro-brain natriuretic peptide (rNT-proBNP), a biomarker of heart failure, in E. coli with high-level productivity. In chapter 4 and 5, I develop a bacterial expression system that produces high yields of soluble growth factors that can be purified in their native forms. The representative growth factors aFGF and VEGF165 were produced. Additionally, keratinocyte growth factor-1 (KGF-1) 135, the most stable form, and KGF-2 were produced by simple processes. Through this research, various human-derived functional healthcare proteins could be produced in E. coli with high quality and high quantity to use the bio-industry.