Microarray analysis of metabolic changes upon co-expressing periplasmic chaperones in scFvD1.3-producing Escherichia coli.
Lim, Denis Yong Xiang.
Date of Issue2009
School of Biological Sciences
High expression of recombinant antibody fragments, such as single-chain variable fragments (scFvs), in Escherichia coli imposes an extra metabolic burden on the cells. This would lead to increased rates of protein mis-folding and accumulation of inclusion bodies, generating stress responses which cause eventual cell death. E. coli periplasmic chaperones skp and fkpA are known to assist in protein folding and assembly. In this study, skp and fkpA were co-expressed in scFv-producing E. coli strains to investigate the effects on soluble scFv yield. DNA microarray studies were performed and gene expression profiles were analyzed to elucidate the underlying differences in metabolic changes of chaperone co-expressing strains as compared to the host strain. Results have shown that skp and fkpA co-expression greatly improved cell viability by 30-70% and scFv-yield by 3-6 folds compared to the wild-type strain. Chaperone co-expressing strains exhibited up-regulation of genes for growth (DNA replication and protein translation) metabolic pathways such as glycolysis and tricarboxylic acid cycle, membrane transporters, cell signaling and motility and down-regulation of genes involved in stress response (heat shock proteins). This represents the microarray global transcriptional analysis of periplasmic chaperone co-expression in scFv-producing E. coli.
DRNTU::Science::Biological sciences::Molecular biology
Final Year Project (FYP)
Nanyang Technological University