Codon Optimisation Is Key for Pernisine Expression in Escherichia coli

Abstract

BackgroundPernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.Methodology/ Principal FindingsThe challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised with a S355A mutation of a predicted active site (pernisineS355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni2+ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisinewt was not expressed in E. coli at detectable levels, while pernisineco and pernisineS355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisineco was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisineS355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.Conclusions/ SignificanceThese data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.

Codon harmonization – going beyond the speed limit for protein expression

“…Multiple publications described heterologous protein yield increase after codon usage improvement. This is of particular interest for vaccine manufacturing process and industrial enzyme production as for example alpha‐amylase, pernisine, and prochymosine …”

“…of particular interest for vaccine manufacturing process [9] and industrial enzyme production as for example alpha-amylase, pernisine, and prochymosine [10][11][12] Rare codons generally correlate with lower levels of cognate tRNA and slower translation rates [13,14]. In some cases, their substitution has been shown to adversely affect the expression levels [15], solubility [16], cotranslational modifications [17] of encoded proteins, and to inhibit the functionality of specific proteins [18,19].…”

Effect of supplementing Moringa oleifera essential oils on milk quality and fatty acid profile in dairy sheep

“…The first method is codon optimization of the target gene. Codon optimization is a key step for the successful expression of protein in E. coli even from a distant host (Snajder et al 2015). Indeed, over the last decade, the use of codon-optimized genes in industrial biotechnology has reduced the cost of protein production, through improved protein expression (Elena et al 2014).…”

Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus

“…It has been demonstrated that pernisine can efficiently degrade cellular and infectious bovine prion proteins [6], which today still present a challenge for decontamination processes, and this represents the main potential industrial application for pernisine. The protein sequence of fully unprocessed pernisine (i.e., prepropernisine) consists of 430 amino acids, which includes a putative signal sequence, as the first 24 amino acids, and a proposed proregion, from amino acids 25 to 92 [7, 8]. Pernisine is active at temperatures from 65 to 115 °C, and retains its catalytic activity across a wide pH range, from 4 to 10 [6].…”

“…At the same time, standard industrial fermentation facilities are not suitable for large-scale production of pernisine, due to its extreme cultivation conditions. Recombinant propernisine from Escherichia coli has similar properties to the wild-type enzyme, after an additional thermal activation step [7]. However, since the recombinant enzyme is located in the intracellular region or in the periplasm, additional downstream operations are required for its isolation and effective temperature pre-treatment.…”