Design and expression of peptide antibiotic hPAB-beta as tandem multimers in Escherichia coli.
Peptide antibiotics are small peptides encoded by organism genomic DNA. They are recognized to play important roles in the innate host defense of most living organisms. The growing resistance of bacteria to conventional antibiotics and the need for discovery of new antibiotics have stimulated great interest in the development of peptide antibiotics as human therapeutics. However, preparation of peptide antibiotics at a large scale is a great challenge in developing these commercial products. In this study, tandem repeat multimers of peptide antibiotic hPAB-beta were designed and the recombinant plasmids containing one to eight copies of hPAB-beta gene were generated. Eight genetic engineered bacteria harboring pQE-hPAB-beta1-8 recombinant were able to express the repetitive hPAB-beta multimers of interest in inclusion bodies, respectively. The expressed proteins could reach 2.6-28% of the total proteins. The hPAB-beta trimer construct was selected out for the subsequent study based on its higher expression level (27.8%), which yields in wet cell weights (3.15+/-0.45 g/l) and the fusion protein inclusion bodies was able to completely dissolve in 8 M urea. The tandem trimers could easily be captured by Ni-NTA affinity chromatography and cleaved into monomers by hydroxylamine. Then, the monomer hPAB-beta of interest was purified to 95% homogeneity by reverse phase chromatography and gel filtration. The final yield of purified recombinant monomer hPAB-beta was 680+/-12 mg/100 g wet cells. The minimum inhibitory concentrations (MICs) of the purified recombinant hPAB-beta against type or clinical strains of microorganisms were about 31-250 microg/ml and these results showed that the recombinant hPAB-beta could retain its bioactivity.
Rao
Xiancai
X
Department of Microbiology, College of Medicine, The Third Military Medical University, Key Lab of Microbial Engineering Under the Educational Committee in Chongqing, Chongqing 400038, PR China.
Hu
Jinchuan
J
Li
Shu
S
Jin
Xiaolin
X
Zhang
Chun
C
Cong
Yanguang
Y
Hu
Xiaomei
X
Tan
Yinling
Y
Huang
Jianjun
J
Chen
Zijin
Z
Zhu
Junming
J
Hu
Fuquan
F
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D020080:Tandem Repeat Sequences; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D010446:Peptide Fragments; D011994:Recombinant Proteins; D023083:beta-Defensins; C503956:hPAB-beta protein, recombinant