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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
Abstract:   (4541 Views)

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.

Keywords: Key words: Glycosylation, Stem cell, PCR
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Type of Study: Research | Subject: Biotechnology
Published: 2017/03/14
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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
URL: http://bloodjournal.ir/article-1-1060-en.html


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Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
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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