Recent contributions in the field of the recombinant expression of disulfide bonded protein in bacteria

Abstract

The production of heterologous disulfide bonded proteins in bacteria remains a biotechnological challenge. A rapid literature survey results in the identification of some interesting proposals, such as the option of producing functional proteins in the cytoplasm in the presence of sulfhydryl oxidases and isomerases. Furthermore, an ever-increasing number of applications refers to recombinant proteins displayed at the bacterial surface. Time will tell whether these developments will lead to universally accepted laboratory protocols.

Bacterial cytoplasm as an effective cell compartment for producing functional VHH-based affinity reagents and Camelidae IgG-like recombinant antibodies

“…We designed a decision chart with the aim of optimizing the cytoplasmic expression of fusion antibodies that either failed to be expressed in the periplasm or, such as the Fc-fusions, accumulated in low amounts (Additional file 2: Figure S2). Bacterial mutants in which the cytoplasmic reducing metabolism is impaired have been sometimes successfully used to express disulfide-dependent proteins but the results are contradictory specially when molecules with multiple disulfide bonds must be produced [11,18]. Therefore, we evaluated the promising alternative of inducing the formation of disulfide bonds in the cytoplasm of wild type bacteria in which a recombinant eukaryotic sulfhydryl oxidase is accumulated [16].…”

Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels

“…Firstly, the isolation of proteins from the periplasm is usually easier than the isolation of proteins from total cell lysates, since the periplasm represents a less complex protein mixture than the cytoplasm [2]. Secondly, the Dsb-system in the periplasm can catalyze the formation of disulfide bonds, whereas the reducing cytoplasm prevents disulfide bond formation [4,5]. To produce heterologous proteins in the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the Sec-translocon.…”

“…It is easier to isolate proteins from this compartment than from whole cell lysates, and, more importantly, in the oxidizing environment of the periplasm the disulfide bond formation (Dsb)-system catalyzes the formation of disulfide bonds. Therefore, disulfide bond containing proteins, like antibody fragments and many peptide hormones, are produced in the periplasm to enable folding into their native conformation [4,6]. …”

Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner

“…Compared with the periplasmic accumulation, Cel-CD can carry the antimicrobial peptides into the culture medium, which enabled the scaled production by overcoming the spatial limitation of periplasmic space. In addition, the secretion of Cel-CD and its recombinant proteins was a two-step process, and the oxidizing environment and Dsb system of periplasm benefits the formation of disulfide bonds (de Marco, 2012).…”