Expression of bioactive recombinant GSLL-39, a variant of human antimicrobial peptide LL-37, in Escherichia coli.
The human cationic antimicrobial peptide hCAP-18/LL-37 is the unique cathelicidin identified in human to date. It has broad spectrum of antimicrobial activities and LPS-neutralizing activity and is involved in angiogenesis. Both purified and synthetic LL-37 or its derivatives were used in the study on LL-37. However, production of LL-37 in Escherichia coli has not been established. In this study, its precursor instead of the mature peptide was adopted for expression to avoid the lethal effect of recombinant LL-37 on host cells. A thrombin recognition site was introduced between the cathelin-like domain and LL-37 domain by overlap PCR to construct fragment encoding modified precursor (mhCAP-18) to facilitate the final release of the recombinant peptide. Then mhCAP-18 was fused in-frame to thioredoxin gene under the control of inducible T7 promoter to construct expression vector pET-mhCAP-18. The soluble form fusion protein was expressed in E. coli and purified by Chelating Sepharose column chromatography. Thrombin digestion of the fusion protein yielded recombinant GSLL-39, which was then purified by cation-exchange chromatography. Recombinant GSLL-39, which has two extra residues on its N-terminus when compared with its native counterpart, showed similar antimicrobial activities against both Gram-negative and Gram-positive bacteria.
State Key Laboratory for Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300020, PR China.
Ying-Hua
YH
Yang
Guo-Guang
GG
Zheng
Ge
G
Li
Xiu-Jun
XJ
Zhang
Zhen-Yu
ZY
Cao
Qing
Q
Rao
Ke-Fu
KF
Wu
D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D054804:Cathelicidins