A plasmid system for optimization of Fab′ production in Escherichia coli: importance of balance of heavy chain and light chain synthesis

Prokaryotic expression of antibodies

“…The complexity and nature of antibody fragments affects expression profiles. For Fabs, factors such as transfer to the periplasm, assembly rate, balance between the synthesis of two chains and the order of Fd (V H + C H 1) and light chain genes are important [98]. For scFv molecules, the length and amino acid composition of the linker can have great impact on expression [21,23,43,99,100].…”

“…Protein yield improvements have been attributed to the formation of favorable 5'-end mRNA secondary structure which enhances translation initiation. The signal peptide wobble-base codon mutagenesis/alkaline phosphatase reporter system was also applied to improving Fab' expression, significantly increasing the yield to 580 mg/L [98,108]; a good balance of light and Fd chain synthesis was found to be important in obtaining higher yield. In another experiment, the same authors [98] increased the expression of the Fab' by 3-fold with a codon usage optimization approach different from above; the 5' codons of the light chain were replaced not with the average preferred codons but with the preferred codons used at the 5' end of E. coli periplasmic genes [15].…”

“…The signal peptide wobble-base codon mutagenesis/alkaline phosphatase reporter system was also applied to improving Fab' expression, significantly increasing the yield to 580 mg/L [98,108]; a good balance of light and Fd chain synthesis was found to be important in obtaining higher yield. In another experiment, the same authors [98] increased the expression of the Fab' by 3-fold with a codon usage optimization approach different from above; the 5' codons of the light chain were replaced not with the average preferred codons but with the preferred codons used at the 5' end of E. coli periplasmic genes [15]. Balanced light and Fd chain synthesis, possibly caused by changes in the secondary structure of the RNA in and around the mutated area may have led to a more favorable secretion/folding process, with the net result being an increase in functional protein expression.…”

Evaluation of the effects and interactions of mixing and oxygen transfer on the production of Fab’ antibody fragments in Escherichia coli fermentation with gas blending

“…Lactose acted as the inducer for Fab' formation and resulting in a reduction of the cell growth. This batch-fed feeding strategy was based on the protocol developed by Celltech Chiroscience Ltd. and is reported elsewhere [8,23]. Assays were carried out on these samples to determine the concentrations of residual substrates, the levels of product formation and the biomass achieved.…”

High-level production of a single chain antibody against anthrax toxin in Escherichia coli by high cell density cultivation

“…In E. coli, large-scale production of recombinant proteins including various antibodies has been successfully accomplished by high cell density cultivation (HCDC) technologies that can achieve cell densities higher than OD 600 of 100 [11,12]. Since Carter et al [13] first reported the production of 1-2 g/L of humanized F(ab) 2 in a 10 L culture of E. coli, HCDC of E. coli has been used for the production of many antibody fragments as well as full length IgG [14][15][16][17][18][19][20]. Horn et al [17] performed the fed-batch fermentation for the production of dimeric miniantibodies against EGF-receptor in 10 L bioreactor, and they could successfully produce more than 3 g/L of soluble antibodies, which is the best reported so far.…”