Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli.
Insect cecropins are small basic polypeptides synthesized in fat body and hemocytes in response to bacterial infections or hypodermic injuries. To explore a new approach for high expression of soluble cecropin in Escherichia coli cells, we fused the sequence encoding Musca domestica mature cecropin (named Mdmcec) in-frame to thioredoxin (TRX) gene to construct an expression vector pTRX-6His-Mdmcec. An enterokinase cleavage site was introduced between the 6xHis-tag and Mdmcec to facilitate final release of the recombinant Mdmcec. The fusion protein TRX-6His-Mdmcec was purified successfully by HisTrap HP affinity column and a high yield of 48.0mg purified fusion protein was obtained from 1L culture. Recombinant Mdmcec was readily obtained by enterokinase cleavage of the fusion protein followed by HPLC chromatography, and 11.2mg pure active recombinant Mdmcec was obtained from 1L E. coli culture. The molecular mass of recombinant Mdmcec determined by electrospray ionization-mass spectrometry (ESI-MS) is identical to that of native cecropin. Analysis of recombinant Mdmcec by circular dichroism (CD) indicated that recombinant Mdmcec contained predominantly alpha-helix with some random coil. Antimicrobial activity assays demonstrated that recombinant Mdmcec had a broad spectrum of activity against fungi, Gram-positive and negative bacteria. The procedure described in this study will provide a reliable and simple method for production of different cationic peptides for biological studies.
Xu
Xiaoxia
X
State Key Laboratory of Biocontrol, College of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China.
Jin
Fengliang
F
Yu
Xiaoqiang
X
Ji
Shunxia
S
Wang
Juan
J
Cheng
Hongxing
H
Wang
Chao
C
Zhang
Wenqing
W
D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D017344:Genes, Insect; D005822:Genetic Vectors; D006461:Hemolysis / Q000187:drug effects; D006793:Houseflies / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins