Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Abstract

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (ϳ33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (ϳ2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.

High and compact formation of baculoviral polyhedrin‐induced inclusion body by co‐expression of baculoviral FP25 in Escherichia coli

“…The nucleotide sequences of the inserted genes were verified by direct sequencing. For confirmation experiments, recombinant plasmid pPAPF that can co-express Polh-Hal18 and FP25 was constructed based on the recombinant plasmid pPAP containing polh-hal18 fusion gene (Wei et al, 2005).…”

“…In addition, the recombinant Polh had almost the same characteristics as the native crystal matrix form of baculoviral polyhedra, including rapid solubilization under alkaline conditions and degradation by polyhedraassociated alkaline proteases. Therefore, using these specific properties of Polh protein, we successfully demonstrated easy and efficient production and/or purification of some target proteins Wei et al, 2005).…”

“…However, it also has several advantages, such as generally resulting in greater expression of foreign protein (about 50% or more of the total cellular protein under appropriate conditions), allowing easy isolation of the protein at high purity (up to 90% purity under optimal conditions) and concentration, effectively protecting the target protein from proteolysis, and allowing efficient production of harmful or toxic proteins (Kane and Hartley, 1988;Lilie et al, 1998;Makrides, 1996). Previously, we showed that AcNPV Polh could be successfully used as a fusion partner for the expression of recombinant proteins (such as green fluorescent protein (GFP), Bacillus thuringiensis (Bt) toxin protein, and antimicrobial peptides) as insoluble inclusion bodies in an E. coli expression system, and that this Polhfusion strategy enabled efficient production of the target proteins (Seo et al, 2003Wei et al, 2005). In addition, the recombinant Polh had almost the same characteristics as the native crystal matrix form of baculoviral polyhedra, including rapid solubilization under alkaline conditions and degradation by polyhedraassociated alkaline proteases.…”

High-Throughput and Facile Assay of Antimicrobial Peptides Using pH-Controlled Fluorescence Resonance Energy Transfer

“…We demonstrated the application of pH-based FRET for quantitative assay of AMPs by investigating the FRET spectrum and Y/C ratio, which is the FI ratio between EYFP at 527 nm and ECFP at 475 nm when emission is triggered from ECFP-EYFP fusion proteins by excitation at 433 nm, for various concentrations of three different proteins: BSA (negative control) and the AMPs halocidin 18-mer (Hal 18) (8,14) and magainin II (16). Although antimicrobial ac- on May 11, 2018 by guest http://aac.asm.org/ tivity is generally evaluated based on g/ml amounts, we utilized the molar concentration in these experiments in an effort to equalize the number of effective molecules, because BSA has a much larger molecular mass (67 kDa) than Hal 18 (1.93 kDa) and magainin II (2.43 kDa).…”

Purification Propensity for Proteins from Bacillus halodurans

“…At protein terminal level, hydrophilicity of histidine tag enhances the high solubility of expressed recombinant fusion proteins [8]. At protein level, polyhedrin is used as a carrier protein to facilitate antigen purification [9][10][11][12][13].…”