pastoris X-33 was 3.7- and 16-fold higher (28.2 μg/ml and 1,024 BU/ml), respectively, than that from the native E. faecium P13 ; in fact, even though the level of 45.1 μg/ml of recombinant enterocin A expressed by P. pastoris  was still too low for its industrial production
and end application, it demonstrates the potential to increase its productivity to be as high as possible and to further easily characterize its purification and properties. However, there are only few studies at the modification of bacteriocin genes, such as gene synthesis or codon optimization, which is considered as a promising technique for increasing protein expression level ; thus, further work with this system is necessary to achieve an increased protein expression level of target Caspase inhibitor selleck screening library gene. Due to the high anti-Lister activity of EntA and its low yield either in native strain and recombinant expression system, the EntA gene was optimized by the preferential codon usage of P. pastoris and was expressed into medium as recombinant EntA (rEntA). The purification of rEntA from ferment supernatant was tried by four methods including gel filtration chromatography, then the antimicrobial activity, proteolytic sensibility and stabilities of heat, pH and salt of purified rEntA were examined. Results Construction and transformation of the expression vector Compared to naturally occurring EntA, the
base codons coding for 37 residues (78.72%) in total 47 amino acids were optimized by the preferential codon usage of P. pastoris (Figure 1A). The GC content of the full target sequence increased from 41.13% to 41.9%. The gene sequence of the optimized EntA was synthesized and inserted into pPICZαA between XhoI and XbaI sites (Figure 1B, C). The expression vector pPICZαA-EntA was transferred into competent E.
coli DH5α cells. Resulting transformants were confirmed by PCR and DNA sequencing. Correct plasmid and control vector pPICZαA were linearized by PmeI and transferred into competent P. pastoris X-33 cells by electroporation. Positive transformations CHIR99021 were screened and confirmed by colony PCR. Figure 1 Construction of the expression plasmid pPICZ α A-EntA. A, The nucleotide sequence of EntA and its corresponding amino acid sequence. The upper line indicates the wild-type EntA gene sequence. The middle line is the codon-optimized EntA gene sequence. Optimized codons are underlined with boldface type. The lower line represents the amino acid sequence of EntA. The termination codon is marked by an asterisk. B, Map of the recombinant plasmid pPICZαA-EntA. C, Electrophoretic analysis of the recombinant vector containing the EntA gene. Lane 1, DNA marker; lane 2, pPICZαA-EntA digested by XhoI and XbaI. Expression of rEntA in shake buy LDN-193189 flasks and at the fermenter level The heterologous expression of rEntA in P. pastoris X-33 was induced by methanol at the concentration of 0.