Enhanced cecropin B2 production via chitin‐binding domain and intein self‐cleavage system

Abstract

In this study, various constructs and hosts were used to produce high levels of cecropin B2 (cecB2). To mitigate cecB2's toxic inhibition of host cells, various cecB2 constructs were built. Results showed that the combination of a chitin-binding domain and an intein self-cleavage motif in front of cecropin B2, without a His-tag, was best for cecB2 expression. E. coli ER2566 was the best host, and 2YT was the best medium for cultivation. Under these conditions, a cecB2 yield of 98.2 mg/L could be obtained after purification. The purified cecB2 expressed a wide antimicrobial effect on most Gram-negative strains, including multidrug-resistant Acinetobactor baumannii and Staphylococcus aureus. This study provides a systematic approach to the efficient production of the antimicrobial peptide (AMP) cecB2 via the recombinant E. coli process, which is expected to be an efficient way for the production of other AMPs.

Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants

“…Prior to the MD simulation, the structural model of CBD-INT-OCVs-cecB2 fusion protein had to be built [17]. Based on the Phyre2 server [41], three structures were applied as templates-the structure of the CBD of chitinase A1 from Bacillus circulans WL-12 (PDB ID: 1ED7), the crystal structure of DnaB INT from Synechocystis sp.…”

“…Some studies reported on the use of microbes as hosts for the production of AMPs [10][11][12][13][14][15][16]; the major concern with this approach is that AMPs could prove toxic to their hosts. While the chitin-binding domain (CBD)-intein (INT) (CBD-INT) domain of pTWIN1 vector (NEB, USA) was helpful in cecB2 production [17], a key issue for this process was how to accelerate the pH-inducible INT self-cleavage reaction. INT self-cleavage is highly dependent on the amino acids adjacent to the INT (the +1 position of the target protein) [18].…”

“…In our previous study, the production of cecB2 peptide was performed with the construction of a fusion protein with CBD-INT [17]. We found that the INT cleavage reaction was the rate-limiting step of the whole process.…”