Design and Unique Expression of a Novel Antibacterial Fusion Protein Cecropin B-Human Lysozyme to Be Toxic to Prokaryoti
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Design and Unique Expression of a Novel Antibacterial Fusion Protein Cecropin B-Human Lysozyme to Be Toxic to Prokaryotic Host Cells Yeli Zhang 1 & Yuhua Li 1 & Liujiao Bian 1
# Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract A novel antibacterial fusion protein, cecropin B-human lysozyme (CB-hLyso), was designed and expressed in a prokaryotic system. The full-length CB gene was first synthesized and fused to the 5′ end of the hLyso gene. The recombinant CB-hLyso was then subcloned in plasmid pET32a, and pET32a-CB-hLyso was transferred into Escherichia coli (E. coli) BL21(DE3) and BL21(DE3)pLysS. The results showed that in the original culture media, Luria-Bertani (LB) media and terrific broth (TB), at 37 or 25 °C, CB-hLyso was barely expressed; however, when the original culture medium was replaced with an equi-volume of fresh medium, obvious expression occurred in BL21(DE3)pLysS/pET32a-CB-hLyso at 25 °C, and the expression in TB (25%) was higher than that in LB (15%). Through a two-step chromatographic method consisting of Ni-chelated Sepharose Fast Flow affinity and Sephadex G-75 size-exclusion, the crude fusion CB-hLyso was isolated in a homogeneous form, and preliminary bacteriostasis experiments showed that the fusion CB-hLyso had a strong inhibitory effect on the growth of Staphylococci. This work provides useful insights into the design of novel fusion polypeptides with higher bacteriolytic activity and wider antimicrobial spectra and in the expression of polypeptide products that are toxic to prokaryotic host cells, eukaryotic host cells or insect cells. Keywords Antibacterial fusion protein . Cecropin B . Human lysozyme . Design . Expression
Introduction The growing resistance of humans, animals, and plants to antibiotics has commanded increasing attention. It has become an urgent task to develop novel active substances to replace antibiotics [18]. Antibacterial peptides (ABPs) are one of these active substances. ABPs are polypeptides from induced organisms and contain 20 to 60 amino acid residues [11]. The uniquely germicidal mechanisms of ABPs make them active against nearly all types of pathogenic bacteria and exhibit a wide antimicrobial spectrum. Currently, more than 100 kinds of different antibacterial peptides have been identified in different animal tissues [7, 10, 12]. Cecropins are one of these natural Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12602-019-09527-8) contains supplementary material, which is available to authorized users. * Liujiao Bian [email protected] 1
College of Life Science, Northwest University, Xi’an 710069, Shaan’xi, PR China
antibacterial peptides and have the highest antimicrobial and antiviral activity. Cecropins usually consist of 37–39 amino acid residues and have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment. Cecropin B (CB), a soluble polypeptide, is one of these cecropins and has a strong resistance to thermal shock and possesses broad antimicrobi
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