Recombinant production, purification and characterization of vessel dilator in E. coli.
Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.
Proteowa Pty Ltd, SABC, Murdoch University, 90 South Street, Murdoch, Western Australia, 6150, Australia; Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
Mahdi
M
Abbasian
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, Iran; Metabolic Disorders Research Center, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Hadieh Alsadat
HA
Eslampanah Seyedi
Department of Biotechnology, College of Agriculture, Isfahan University of Technology, Isfahan, 84156-83111, Iran.
Badraldin Ebrahim
BE
Sayed Tabatabaei
Metabolic Disorders Research Center, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran; Department of Biochemistry and Biophysics, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
Zahra
Z
Arab-Bafrani
Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran.
Mohammad Reza
MR
Mofid
Proteowa Pty Ltd, SABC, Murdoch University, 90 South Street, Murdoch, Western Australia, 6150, Australia. Electronic address: rzareie@proteowa.com.au.
Reza
R
Zareie
D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000096:biosynthesis / Q000302:isolation & purification; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D000970:Antineoplastic Agents; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C054548:atrial natriuretic factor prohormone (31-67); C033223:polyhistidine; D006639:Histidine; D009320:Atrial Natriuretic Factor
Expression prediction; Overexpression; Tandem peptides; Vessel dilator