Molecular cloning and expression of ranalexin, a bioactive antimicrobial peptide from Rana catesbeiana in Escherichia coli and assessments of its biological activities.
The coding sequence, which corresponds to the mature antimicrobial peptide ranalexin from the frog Rana catesbeiana, was chemically synthesized with preferred codons for expression in Escherichia coli. It was cloned into the vector pET32c (+) to express a thioredoxin-ranalexin fusion protein which was produced in soluble form in E. coli BL21 (DE3) induced under optimized conditions. After two purification steps through affinity chromatography, about 1 mg of the recombinant ranalexin was obtained from 1 L of culture. Mass spectrometrical analysis of the purified recombinant ranalexin demonstrated its identity with ranalexin. The purified recombinant ranalexin is biologically active. It showed antibacterial activities similar to those of the native peptide against Staphylococcus aureus, Streptococcus pyogenes, E. coli, and multidrug-resistant strains of S. aureus with minimum inhibitory concentration values between 8 and 128 μg/ml. The recombinant ranalexin is also cytotoxic in HeLa and COS7 human cancer cells (IC50 = 13-15 μg/ml).
Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
Rasha Abou
RA
Aleinein
Razan
R
Hamoud
Holger
H
Schäfer
Michael
M
Wink
D000818:Animals; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D019556:COS Cells / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D011892:Rana catesbeiana / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D013211:Staphylococcus aureus / Q000187:drug effects; D013297:Streptococcus pyogenes / Q000187:drug effects
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; C086618:ranalexin