Cloning and Expression of recombinant human midkine gene in Escherichia coli origami
Volume 6, Issue 2, Summer 2015, Pages 143-151
https://doi.org/10.52547/JCT.6.2.143
S Gh, A D, A B, E E, B M, F R
Abstract Aim: The aim of this research was cloning and expression of human Midkine coding gene (mdk) in Escherichia coli that achieved in a laboratory-scale experiment.
Material and Methods: Methods were included cell culture, RNA extraction, cDNA synthesis, cloning techniques, induction of expression by IPTG (isopropyl thiogalactosidase), expression evaluation using polyacrylamide gel and confirmation by Western blot techniques.
Results: Midkine gene was cloned in pET-21a (+) and then transformed into Origami strain of E. coli. This growth factor was expressed in cytoplasmic level by a colony containing pETmdk recombinant after 16 hours incubation at 18°C and 250 rpm mixing. Expression of histidine tagged 13 kD protein confirmed by Western blotting technique.
Conclusion: Because Origami strain is trxB and gor genes mutant strain its cytoplasm is an oxidizing environment. Due to this, it enhances disulfide bond forming, therefore it seems that after expression of midkine, cysteine residues make an intra-molecular disulfide bridge and remains in soluble form. These conditions provide suitable environment for the proper folding of the protein and consequently solubilization of the protein.