Bioproduction and characterization of a pH responsive self‐assembling peptide

Abstract

Peptide P(11)-4 (QQRFEWEFEQQ) was designed to self-assemble to form beta-sheets and nematic gels in the pH range 5-7 at concentrations > or =12.6 mM in water. This self-assembly is reversibly controlled by adjusting the pH of the solvent. It can also self-assemble into gels in biological media. This together with its biocompatibility and biodegradability make P(11)-4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large-scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P(11)-4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P(11)-4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto-induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C-terminal homoserine lactone (rP(11)-4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed beta-structure measured by circular dichroism and fibril formation observed by transmission electron microscopy.

A self-assembling peptide acting as an immune adjuvant

“…The peptide Q11 was previously designed as a self-assembling transglutaminase substrate (22) and was a variation on the DN1 peptide originally described by Aggeli and coworkers (25,33). For the work…”

“…The peptide Q11 was previously designed as a self-assembling transglutaminase substrate (22) and was a variation on the DN1 peptide originally described by Aggeli and coworkers (25,33). For the work reported here, we designed a peptide containing a Q11 selfassembling domain in tandem with OVA 323-339 , a 17-amino acid peptide from chicken egg ovalbumin (Fig.…”

Bioproduction and self-assembly of designer peptides

“…One effective approach used to increase the proportion of target peptide in the expressed product is the incorporation of multiple copies of the target sequence. [310][311][312][313] However, the number of incorporated repeats cannot be increased indefinitely, and the higher repeat number constructs tend to display lower expression. [310][311][312][313] The optimal repeat numbers for expression must therefore be determined empirically for each expression system.…”

“…The majority of published methods for the purification of expressed peptides utilise reversed phase, affinity or ion-exchange chromatography, either in single or sequential steps. 274,306,311,312,317,[341][342][343] This is due to the high level of purity attained with these methods as well as technique simplicity and adaptability for varied peptides. While these methods are effective at the laboratory scale for early peptide characterisation, chromatography-based approaches add significant cost to overall peptide production and are impractical for large-scale processes.…”

“…349 Several of these biologically produced peptides display altered self-assembly compared to synthetically produced equivalents. 306,311,349 These examples illustrate the potential for sequence modifications to affect peptide properties. Given the limited range of cleavage methods available, it seems likely that compatibility of peptide sequences with these methods will be a limiting factor in determining sequences that are amenable to bioproduction.…”