Engineering of escherichia coli for the enhanced production of recombinant antibodies

Abstract

Currently, antibodies that play a major role in treating a wide variety of human diseases (e.g., cancer, viral infection, inflammation) have become major therapeutic reagents in the pharmaceutical drug market. In addition to full-length antibodies, the development of antibody fragments, which also offers potential ad-vantages in clinical use as well as diagnostics, is gradually growing. As the demand for antibody therapeutics increase, the development of host systems for enhanced and less expensive production has also become more important. Most of antibody therapeutics approved to date is predominantly produced in mammalian hosts, but due to drawbacks such as high production cost and long-term cultivation, the alternative use of bacteria has been seriously considered. Among various bacteria, Escherichia coli is one of the most feasible host for antibody production, because of its well-known and robust features. Indeed, E. coli has been widely utilized for the production of antibody fragments, but in many cases, it has been frequently faced with the limitations of low production yield and secretion efficiency. Moreover, production of full-length antibody has been regarded as more difficult problem due to the large size, complex structure and lack of glycosylation. In this study, to suggest generic solutions for those problems, various strategies were examined and inten-sively studied. Firstly, to facilitate the secretion of antibodies in E. coli, SRP-dependent secretion pathway, instead of conventional Sec-dependent pathway, was utilized and engineered. The SRP pathway has a great feature of co-translational translocation, thus it was expected that the cytoplasmic aggregation of antibodies could be prevented. Indeed, expression level of scFv and IgG was increased by simple changing of secretion pathway (using SRP instead of Sec). In addition to the utilization of SRP pathway, co-expression of the SRP pathway-related factors (i.e., ffh, ftsY, yidC) and/or folding assistants (i.e., dsbC, dsbA, skp) was conducted to improve the secretion and folding efficiency. As a result, scFv and IgG were increasingly produced through the SRP pathway in E. coli. Secondly, to lead the significant increase of antibody expressions, two kinds of cellular engineering were conducted. To facilitate binding between ribosome and mRNA, 5’ and/or intergenic untranslated region (UTR) sequences were modified and the effects were investigated. In addition, single gene (rnpA) knockdown, which offers higher stability of antibody-encoding mRNA, was introduced. For the results of the both strate-gies, production yields of IgG were dramatically increased. Moreover, knockdown of rnpA also led the en-hanced production of Fab fragment. Finally, to improve the performance of SRP-dependent pathway in E. coli, I developed a novel E. coli mu-tant through the FACS-based directed evolution technique. For this experiment, I developed a new fluores-cent reporter system, which can visualize the periplasmic expression level of target protein, by using a special fluorescent dye, FlAsH-EDT2. Using these strategies, transposon-mediated E. coli mutant library was screened, and so, single copy of 16S rRNA (rrsE) knock out mutant was isolated. With this mutant, several model proteins (i.e., MBP, DsbA, TorT, TolB) were increasingly expressed and secreted through the SRP pathway. Moreover, the production yield of antibodies (i.e., IgG, scFv) and G protein-coupled receptor were dramatically increased since the great effect of isolated mutant.