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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

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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.

Schematic representation of expression vector pET-32a-CB-hLyso, with Factor Xa and Asn-Gly.

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Funding

This work was supported by a grant from the National Natural Science Foundation of China (Grant 21075097).

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Authors and Affiliations

  1. College of Life Science, Northwest University, Xi’an, 710069, Shaan’xi, PR China

    Yeli Zhang, Yuhua Li & Liujiao Bian

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  1. Yeli Zhang
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  2. Yuhua Li
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  3. Liujiao Bian
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Correspondence to Liujiao Bian.

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Zhang, Y., Li, Y. & Bian, L. Design and Unique Expression of a Novel Antibacterial Fusion Protein Cecropin B-Human Lysozyme to Be Toxic to Prokaryotic Host Cells. Probiotics & Antimicro. Prot. 11, 1362–1369 (2019). https://doi.org/10.1007/s12602-019-09527-8

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  • DOI: https://doi.org/10.1007/s12602-019-09527-8

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