Promoter engineering to optimize recombinant periplasmic Fab′ fragment production in Escherichia coli

Abstract

Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016.

Engineering and application of synthetic nar promoter for fine-tuning the expression of metabolic pathway genes in Escherichia coli

“…The reporter gfpm gene was amplified by PCR from pQE- gfpm [ 22 ] with primers ( Xma I-gfpm-F and Not I-gfpm-R) and cloned downstream of the lac promoter of pUCM, generating pUCM-gfpm. To obtain pSTVM- gfpm , the PCR-amplified gfpm gene containing the lac promoter was inserted between the Bam HI and Eco RI sites of the pSTVM2 plasmid [ 12 ]. The ldhD -encoding d -lactate dehydrogenase of Leuconostoc citreum , ilvBN -encoding acetohydroxy acid synthase of E. coli , aldB -encoding acetolactate decarboxylase of Lactococcus lactis , and bdh1 -encoding butanediol dehydrogenase from Saccharomyces cerevisiae were amplified by PCR from the genomic DNAs of each strain, and then cloned downstream of the synthetic nar promoters of pUCNrS, pUCNrI, and pUCNrW (Table 2 ).…”

“…Among them, promoter engineering has been proposed as one of the most efficient ways of fine-tuning transcriptional control in Escherichia coli , Corynebacterium glutamicum , Bacillus subtilis , and yeasts [ 3 – 10 ]. For example, the E. coli strain with engineered l -phenylalanine-responsive promoter could produce fourfold higher titer of phenylalanine than wild-type promoter [ 11 ], and the engineered tac promoter library could decrease leakage of antibody fragment expression in E. coli [ 12 ].…”

Strategies and Applications of Antigen-Binding Fragment (Fab) Production in Escherichia coli

“…Schofield et al reduced Fab′ leakage by converting the promoter from Ptac to Ptic using site-directed mutagenesis. 61 Balancing promoter strength with the overall metabolic burden of cellular "housekeeping" processes could also address translocon overload, caused by periplasmic translocation of exogenous proteins. Wacker's ESETEC secretion technology was an efficient technology for the expression and secretion of Fab exceeding 4.0 g/L, which was approximately 40-fold higher than secretion into the periplasm.…”

“…Fab fragments are classically generated by digesting immunoglobulin with papain, and purified by protein L affinity chromatography with a strong affinity to the variable region of kappa Lc of IgG molecule. 61 However, its applicability is limited to the kappa light-chain Fab subclass. Furthermore, protein L requires extreme pH for elution and its performance is often not comparable with protein A in the binding affinity, selectivity, and capacity.…”

A novel strategy for efficient expression of an antibody fragment in Escherichia coli: ranibizumab as a case study

“…Recombinant protein expression with periplasmic signal sequences has been reported to result in leaky expression. 9 Promoter engineering from Ptac to Ptic (two nucleotides added between −10 and −35) has been utilized to address this issue, but this ultimately decreased the Fab expression. Decreasing Fab expression by promoter engineering resulted in a decreased cytotoxic burden on the cell, while maintaining growth and productivity performance.…”