Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins

Abstract

Metallocarboxypeptidase inhibitors are proteins with possible applications in biomedicine given their properties as anticoagulant and antitumoral factors. They are small, eukaryotic polypeptides comprising several disulfide bridges, which makes them hard to express in inexpensive bacterial hosts. In this work, three of them were produced in high-cell-density cultures of Escherichia coli: PCI (39 residues and three bridges), LCI (66 residues and four bridges) and TCI (75 residues and six bridges). The genes coding for the mentioned inhibitors were cloned in an arabinose-inducible plasmid fused to the signal peptide of DsbA in order to have them secreted and grant the formation of the bridges. The trigger-factor defective strain KTD101 was used as the expression host. The resulting recombinant strains were cultured in fed-batch mode employing minimal media and an exponential feed profile, keeping the specific growth rate at μ = 0.1 h(-1) by limitation of the fed carbon source (glycerol). Between 380 and 540 mg l(-1) of active inhibitors were obtained in both the periplasmic extracts and extracellular media of the cultures. Later on, excretion was enhanced using a cell permeabilization treatment, allowing the recovery of over 80% of the products from the extracellular fraction. Protein yields were found to be inversely proportional to cysteine content of the inhibitor, whereas protein excretion rates were inversely proportional to the protein size. Overall, these results offer insight into the secretory production of active disulfide-bridged proteins in high-cell-density cultures of E. coli.

Antifungal Plant Defensins: Mechanisms of Action and Production

“…In both cases, an eukaryotic system is preferred [112]. In addition, Puertas and colleagues reported that the protein yield, when using E. coli as a host for recombinant production, is inversely proportional to the cysteine content of the protein [113]. This indicates the need for other expression systems when producing proteins with a high cysteine content, such as plant defensins.…”

Harnessing the potential of bacterial oxidative folding to aid protein production

“…The DSB system is fundamental for bacterial physiology as it safeguards the homeostasis of the cell envelope proteome (Dutton et al., 2008; Vertommen et al., 2007). Concurrently, these folding catalysts have been invaluable tools for the production of proteins that were unattainable through mainstream purification strategies (Li et al., 2013; O’Reilly et al., 2014; Puertas et al., 2011; Suzuki et al., 2012). This has been achieved through in‐depth understanding of the biochemistry of both the DSB system and of each protein of interest and extensive trial and error.…”

“…Unfortunately, the efficacy of each signal peptide cannot be preempted and must be determined experimentally by trial and error (Selas Castiñeiras et al., 2018). The DsbA SS outperforms other signal peptides for the recombinant expression of several proteins, such as Fbs1 (Chen & Samuelson, 2016), roGH (Durrani et al., 2015) and hGH (Soares et al., 2003), mPRL (Suzuki et al., 2012), as well as potato and tick carboxypeptidase inhibitors (Puertas et al., 2011). Moreover, it has been used for the secretion of engineered single‐domain antibodies, such as scFvs, VNARs, and VHHs, with mixed success (Fink et al., 2019; Kasli et al., 2019; Leow et al., 2019; Monegal et al., 2012; Schofield et al., 2017; Thie et al., 2008).…”

Synthesis, Structure, and Activity of the Antifungal Plant Defensin PvD1

“…Direct isolation from plant extracts usually results in very low yields, a limitation also associated with recombinant expression in prokaryotes. The presence of disulfide bonds escalates the challenge due to the high probability of generation of misfolded structures. , Low availability can be partially circumvented by using yeast expression systems (e.g., Pichia pastoris), which retain the features of higher eukaryotes and provides larger peptide amounts and improved folding compared to bacteria. , Direct secretion of overexpressed protein by yeast to culture media is an additional advantage . However, these systems are associated with frequent incorporation of unintended amino acids at the peptide N-terminus, which are likely to change the biological function and increase the complexity of downstream processing. ,− Thus, potential cost-related advantages of recombinant expression systems apply only to large-scale, optimized production of peptides devoid of posttranslational modifications.…”