ORIGINAL ARTICLES   

Minerva Biotecnologica 2016 March;28(1):1-6

Copyright © 2016 EDIZIONI MINERVA MEDICA

lingua: Inglese

Synthesis, cloning and sequencing of strep tag II-mini human insulin in E. coli

Kafeel AHMAD

Center of Biotechnology and Microbiology, University of Peshawar, KPK, Pakistan

BACKGROUND: Insulin is a hormone that is involved in carbohydrate metabolism. Deficiency of insulin results in diabetes mellitus which is a severe metabolic disorder. With view of the increasing demands for insulin in the near future and to expand the range of modified insulin types to meet the need of diversified patient population, a modified form of human insulin was synthesized and cloned into E coli.
METHODS: The synthetic mini-insulin gene was constructed in vitro as a strep tag ІІ-mini-insulin gene fusion molecule by attaching a strep tag ІІ sequence to the 5′ end of mini-insulin gene. The strep tag ІІ -mini-insulin gene fusion molecule was constructed by designing four partially overlapping oligonucleotides. Oligonucleotides KA001and KA002 were designed to form the 5′ end of the gene fusion molecule and oligonucleotides KA003 and KA004 were designed to form the 3′ end of the gene fusion molecule. Oligonucleotides KA001and KA002 were annealed and extended to form the 5′ end of the gene fusion, while oligonucleotides KA003 and KA004 were annealed and extended to form the 3′ end. The PCR amplified strep-tag II-mini-insulin fusion gene was cloned into pGEM-T easy vector to generate vector pGEMKA1. pGEMKA1 was transformed into Stratagene XL1-Blue cells by heat shock transformation. The gene construct was sequenced for confirmation of correct sequence of the synthesized fusion gene.
RESULTS: The correct sequence of the predicted synthetic insulin was confirmed through DNA sequencing. The synthesized insulin was similar in sequence to the DesB30 form of insulin that is a precursor of pharmaceutical insulin formulations.
CONCLUSION: The newly synthesized insulin construct could be utilized for plant transformation to investigate in planta expression of insulin as cheaper and easily available source of this important pharmaceutical.