Harnessing the potential of bacterial oxidative folding to aid protein production

Abstract

Recombinant protein production is a cornerstone of research and key to the development of diagnostics and therapeutics. From the advent of recombinant insulin production in 1982, protein expression and purification techniques have continuously improved. On average, the FDA has approved 44 protein-based therapeutics per year for the past 5 years, including monoclonal antibodies for the treatment of disease, growth factors, thrombolytics, and immunomodulators (FDA, 2020). These join a growing list of enzymes, reagents, and food additives that aid industrial and academic processes alike (Arbige et al., 2019;Pham, 2018). For the expression of these proteins, non-native expression systems offer unparalleled control on the production process compared to endogenous protein extraction protocols. This is because the user is able to tailor the cultivation parameters for each protein target by adjusting variables like the media composition, pH, agitation, aeration, temperature, cell density, inducer concentration, induction time, and feeding strategy (Ahmad et al., 2018). In particular, bacterial expression systems benefit from short doubling times, inexpensive media, genetic tractability, and ease of scaling up. Nonetheless, most bacterial laboratory strains lag behind eukaryotic expression systems in their ability to efficiently introduce posttranslational modifications (PTMs) onto a polypeptide backbone, and as such they are rapidly falling out of use. Indeed, during the 2014-2018 period, the use of non-mammalian systems for protein expression saw the largest drop on record to

Heterologous expression and characterization of novel GH12 β-glucanase and AA10 lytic polysaccharide monooxygenase from Streptomyces megaspores and their synergistic action in cellulose saccharification

“…Sm Bglu12A and Sm Lpmo10A were initially expressed as inclusion bodies in the E. coli Transetta (DE3), which might be attributed to the reducing environment of bacteria inhibiting the correct formation of disulfide bridges [ 23 ]. Based on several strategies that had been developed in recent years to improve the ability of disulfide linkages formation [ 24 , 25 ], the co-expression of different chaperones plasmids in various host strains of E. coli was attempted to achieve soluble expression of Sm Bglu12A and Sm Lpmo10A.…”

Cysteine Redox Chemistry in Peptide Self-Assembly to Modulate Hydrogelation

“…Controlling the correct formation of disulfide bonds to produce functional proteins in vitro is not trivial. Several approaches have been developed to master oxidative folding of recombinant proteins and peptides [ 28 , 29 ]. It is thus not surprising that bioinspired approaches that aim to exploit thiol oxidation to disulfides to control peptide assembly have appeared in the literature in the past in relatively modest numbers.…”