Heterologous expression and purification of dermaseptin S4 fusion in Escherichia coli and recovery of biological activity.
Heterologous expression of dermaseptin S4 (DS4), which has cytolytic activity in vitro against a broad spectrum of pathogenic microorganisms, was examined in Escherichia coli. The plasmid pGEX-4T-1, encoding DS4 fused with glutathione S-transferase (GST), was constructed and cloned into the E. coli strain BL21 (DE3). The fusion protein was overexpressed in this strain after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified to homogeneity using GST affinity chromatography. To recover biologically active DS4, the purified fusion protein was cleaved using thrombin protease; the liberated DS4 was shown to be bactericidally active against an indicator strain. Since it is less expensive to obtain such a peptide biologically, in this study, we report for the first time a method to express purify DS4 in E. coli using a GST fusion system.
a Key Laboratory for Cell and Gene Engineering of Zhejiang Province , College of Life Sciences, Zhejiang University , Hangzhou , China.
Dafeng
D
Song
Yue
Y
Chen
Xuan
X
Li
Muyuan
M
Zhu
Qing
Q
Gu
D029845:Amphibian Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D001001:Anura / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D016982:Micrococcus luteus / Q000187:drug effects / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D013917:Thrombin / Q000737:chemistry
D029845:Amphibian Proteins; D023181:Antimicrobial Cationic Peptides; D011993:Recombinant Fusion Proteins; C070905:dermaseptin; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase; D013917:Thrombin