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:: Volume 14, Issue 1 (Spring 2017) ::
Sci J Iran Blood Transfus Organ 2017, 14(1): 53-64 Back to browse issues page
Isolation, cloning and expression of human stem cell factor using prokaryotic expression system 
M. Nayebhashemi , M. Mohammadi pour , H. Fahimii , M. Habibi Roudkenar , Mohammad Ali Jalili
High institute for research and Education in Transfusion Medicine
Keywords: Key words: Glycosylation, Stem cell, PCR
Full-Text [PDF 492 kb]   (2552 Downloads)     |   Abstract (HTML)  (4570 Views)
Type of Study: Research | Subject: Biotechnology
Published: 2017/03/14
Full-Text:   (4388 Views)
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Original  Article
 

 
 
 
Sci J Iran Blood Transfus Organ 2017; 14(1): 53-64
 
Isolation, cloning and expression of human stem cell
factor using prokaryotic expression system
 
Nayebhashemi M.1, Mohammadipour M.1, Fahimi H.2, Habibi Roudkenar M.3, Jalili M.A.1
 
 
 
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
2Faculty of Advanced Sciences and Technology, Pharmaceutical Sciences Branch of Islamic Azad University (IAUPS), Tehran, Iran
3Biotechnology Research Center, Guilan University of Medical Sciences, Rasht, Iran
 
 
Abstract
Background and Objectives
Stem cell factor (SCF) is a 28-40 kDa glycoprotein which plays an important role for the proliferation and differentiation of hematopoietic stem cells (HSCs). SCF binds to the C-kit receptor, and improves the survival of HSCs in vitro. SCF is one of the essential supplements for cultivation of HSCs in vitro. It plays a vital role to increase the number and size of HSC colonies.
 
Materials and Methods
In this experimental study, glycosylation of SCF does not seem to influence its biological activity; therefore, it would be possible to produce it in prokaryotic expression system. The aim of this study was isolation, cloning and expression of SCF in the Rosetta expression host. In addition to the features of prokaryotic expression system, Rosetta was expected to provide rare codons of eukaryotic proteins and increase the recombinant protein expression level.
Results
The SCF coding sequence was isolated and amplified using the specific primers and cloned in E.coli TOP10 using pET-32a expression vector. The recombinant construct was confirmed by PCR, digestion and sequencing. The recombinant vector was transformed to the expression host, Rosetta.
 
Conclusions
The SCF coding sequence was successfully isolated and cloned into the expression vector pET-32a, followed by recombinant protein expression in Rosetta host strain.
 
Key words: Glycosylation, Stem cell, PCR
 
 
 
 
Received: 14 Aug 2016
Accepted:15 Nov 2016
 
 
 

Correspondence: Jalili MA., PhD of Medicinal Chemistry. Assistant Professor of Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran. Tel: (+9821) 82052155; Fax: (+9821) 88601599
E-mail: m.jalili@ibto.ir
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Nayebhashemi M, Mohammadi pour M, Fahimii H, Habibi Roudkenar M, Jalili M A. Isolation, cloning and expression of human stem cell factor using prokaryotic expression system . Sci J Iran Blood Transfus Organ 2017; 14 (1) :53-64
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Volume 14, Issue 1 (Spring 2017) Back to browse issues page
فصلنامه پژوهشی خون Scientific Journal of Iran Blood Transfus Organ
The Scientific Journal of Iranian Blood Transfusion Organization - Copyright 2006 by IBTO
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