Expression and purification of antimicrobial peptide AP2 using SUMO fusion partner technology inEscherichia coli

Abstract

Significance and Impact of the Study: AP2, an artificial mutant apidaecin (AP) peptide based on APs, has improved activities and may be regarded as a promising antibiotic alternatives. The secreted expression of antimicrobial peptide is of the greatest challenges because the antibacterial activity is not beneficial to the host. Our data suggest that the recombinant fusion strategy allows convenient high yield and easy purification of recombinant AP2. AbstractApidaecins (APs) are proline-rich antimicrobial peptides that were isolated from Apismelifera. APs possess broad-spectrum activities against Gram-negative bacteria and exhibit immune-modulatory functions. AP2, an artificial mutant AP peptide with improved activities, was expressed in Escherichia coli expression system using small ubiquitin-related modifier (SUMO) fusion technology and ZYM-5052 auto-induction medium. Approximately 23 mg of recombinant fusion protein smt3AP2 was purified per litre cultivated medium. After SUMO protease (Ulp) cleavage of smt3AP2, recombinant AP2 was further purified by affinity and cation exchange chromatography. The pure recombinant AP2 with calculated value of 2Á23 kDa reached a yield of 2Á7 mg l À1 and exhibited powerful antibacterial activity towards E. coli K88 with minimum inhibitory concentration at 5 lg ml À1 . The recombinant fusion strategy presented in this study allows convenient high yield and easy purification of recombinant AP2.

Charting the sequence-activity landscape of peptide inhibitors of translation termination

“…Because previous efforts for expressing Api1b in bacteria involved its fusion to longer proteins or focused on minimizing its toxicity (24,25,27,29), we first tested whether the native PrAMP generated endogenously in Escherichia coli cells would retain its antibacterial activity and mechanism of action. We engineered two plasmid constructs containing synthetic api genes, under the control of the arabinose-inducible P BAD promoter, starting with the initiator AUG codon, followed by the codon-optimized sequence specifying the 18 amino acids of Api1b and ending with either UAG (pApi-UAG) or UGA (pApi-UGA) stop codons, decoded by RF1 or RF2, respectively (Fig.…”

Fabrication and characterization of dextran/zein hybrid electrospun fibers with tailored properties for controlled release of curcumin

“…Incorporating curcumin into biopolymer-derived nanofibers may enhance its bioavailability at targeted sites with controlled release. 26,27 The release of curcumin from loaded electrospun fibers can be controlled by modulating the hydrophobicity/ hydrophilicity character of the fibers. 28 In this study, we aimed to fabricate dextran/zein electrospun fibers and investigate the effects of various zein concentrations on the properties of the hybrid electrospun fibers.…”

Efficient and Robust Preparation of Tyrosine Phosphorylated Intrinsically Disordered Proteins

“…The CTD is difficult to produce as IDPs are prone to proteolysis and aggregation [34,65–67]. Several studies have shown improvement in solubility and stability by fusing IDPs to a fusion partner, for example, SUMO [68,69], GST [70] or MBP [9]. We used the SUMO tag, which facilitates expression, solubility and subsequent purification with highly active and site-specific Ulp1 protease [37–39].…”