CLONING AND EXPRESSION OF HUMAN RECOMBINANT MIDKINE GENE IN ESCHERICHIA COLI ORIGAMI STRAIN

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GAFARI S.; DELDAR A.A.; BAHRAMI A.; ESMAEILZADEH E.; MAHYAD B.; ROOHOLLAH F.

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Journal: JOURNAL OF CELL & TISSUE Year:2015 | Volume:6 | Issue:2 Page(s): 143-151

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Title

CLONING AND EXPRESSION OF HUMAN RECOMBINANT MIDKINE GENE IN ESCHERICHIA COLI ORIGAMI STRAIN

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Keywords

ESCHERICHIA COLIQ2

EXPRESSIONQ1

RECOMBINANT PROTEINQ2

CLONINGQ2

MIDKINEQ2

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 18oC 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.

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APA: Copy

GAFARI, S., DELDAR, A.A., BAHRAMI, A., ESMAEILZADEH, E., MAHYAD, B., & ROOHOLLAH, F.. (2015). CLONING AND EXPRESSION OF HUMAN RECOMBINANT MIDKINE GENE IN ESCHERICHIA COLI ORIGAMI STRAIN. JOURNAL OF CELL & TISSUE, 6(2), 143-151. SID. https://sid.ir/paper/188911/en

Vancouver: Copy

GAFARI S., DELDAR A.A., BAHRAMI A., ESMAEILZADEH E., MAHYAD B., ROOHOLLAH F.. CLONING AND EXPRESSION OF HUMAN RECOMBINANT MIDKINE GENE IN ESCHERICHIA COLI ORIGAMI STRAIN. JOURNAL OF CELL & TISSUE[Internet]. 2015;6(2):143-151. Available from: https://sid.ir/paper/188911/en

IEEE: Copy

S. GAFARI, A.A. DELDAR, A. BAHRAMI, E. ESMAEILZADEH, B. MAHYAD, and F. ROOHOLLAH, “CLONING AND EXPRESSION OF HUMAN RECOMBINANT MIDKINE GENE IN ESCHERICHIA COLI ORIGAMI STRAIN,” JOURNAL OF CELL & TISSUE, vol. 6, no. 2, pp. 143–151, 2015, [Online]. Available: https://sid.ir/paper/188911/en

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