Limits...
Expression of Recombinant Human Insulin-like Growth Factor Type 1 (rhIGF-1) in Escherichia coli.

Iranpoor H, Omidinia E, Vatankhah V, Gharanjik V, Shahbazi M - Avicenna J Med Biotechnol (2015 Jul-Sep)

Bottom Line: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells.Molecular weight of the expressed protein was estimated to be 7.6 kDa. hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified.In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Faculty of Advanced Technologies in Medicine, Golestan University of Medical Science, Gorgan, Iran.

ABSTRACT

Background: Human insulin-like growth factor type 1 (hIGF-1) is a protein consisting of 70 amino acids (MW=7.6 kDa) and mainly synthesized by liver. Mecasermin (Trade name INCRELEX) is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli (E. coli) BL21 (DE3) expression system in order to produce an active recombinant form of the protein.

Methods: For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 (DE3) cells through CaCl 2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures.

Results: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa.

Conclusion: hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.

No MeSH data available.


Related in: MedlinePlus

Colony PCR of IGF-1 sequence integrated into pET-24a. Lane 1 contained DNA Ladder (Sinagene (Iran)) and lane 2 contained IGF-1 sequence or the region located between two primers (425 bp band).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4508332&req=5

Figure 2: Colony PCR of IGF-1 sequence integrated into pET-24a. Lane 1 contained DNA Ladder (Sinagene (Iran)) and lane 2 contained IGF-1 sequence or the region located between two primers (425 bp band).

Mentions: In order to confirm ligation of the gene into expression plasmid (pET-24a), colony PCR was run. As it is shown (Figure 2), lane 2 contained IGF-1 sequence and the region was located between two primers (425 bp band).


Expression of Recombinant Human Insulin-like Growth Factor Type 1 (rhIGF-1) in Escherichia coli.

Iranpoor H, Omidinia E, Vatankhah V, Gharanjik V, Shahbazi M - Avicenna J Med Biotechnol (2015 Jul-Sep)

Colony PCR of IGF-1 sequence integrated into pET-24a. Lane 1 contained DNA Ladder (Sinagene (Iran)) and lane 2 contained IGF-1 sequence or the region located between two primers (425 bp band).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4508332&req=5

Figure 2: Colony PCR of IGF-1 sequence integrated into pET-24a. Lane 1 contained DNA Ladder (Sinagene (Iran)) and lane 2 contained IGF-1 sequence or the region located between two primers (425 bp band).
Mentions: In order to confirm ligation of the gene into expression plasmid (pET-24a), colony PCR was run. As it is shown (Figure 2), lane 2 contained IGF-1 sequence and the region was located between two primers (425 bp band).

Bottom Line: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells.Molecular weight of the expressed protein was estimated to be 7.6 kDa. hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified.In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biotechnology, Faculty of Advanced Technologies in Medicine, Golestan University of Medical Science, Gorgan, Iran.

ABSTRACT

Background: Human insulin-like growth factor type 1 (hIGF-1) is a protein consisting of 70 amino acids (MW=7.6 kDa) and mainly synthesized by liver. Mecasermin (Trade name INCRELEX) is the synthetic form of the protein which is used as an effective treatment for particular disorders such as short stature, type 1 and 2 diabetes, and wound healing. Current study was aimed to investigate the expression of human insulin-like growth factor type1 in Escherichia coli (E. coli) BL21 (DE3) expression system in order to produce an active recombinant form of the protein.

Methods: For the purpose of the study, firstly codon optimization was done for hIGF-1 gene, using bioinformatics databases. Then, the gene was synthesized and inserted in pET-24a vector by a cutting strategy included NdeI and BamHI-HF enzymes. In the next step, gene was run in agarose gel and purified. The constructed expression cassette was transformed into E. coli BL21 (DE3) cells through CaCl 2 heat shock method. Identification and confirmation of the transformed colonies were performed using screening PCR method. Synthesis of hIGF-1 was induced by IPTG. The expression in induced strains was analyzed by SDS-PAGE and western blotting techniques. Confirmation of cloning and IGF-1 expression cassette was carried out through genetic engineering procedures.

Results: Analysis of transformed E. coli strain with SDS-PAGE and western blotting techniques confirmed that gene was expressed in host cells. Molecular weight of the expressed protein was estimated to be 7.6 kDa.

Conclusion: hIGF-1 expression cassette for cloning and expression in E. coli was designed and the protein of interest was successfully induced and identified. In addition, E. coli BL21 (DE3) can be used as a suitable host for production of recombinant hIGF-1 and this technology has a potential to be localized.

No MeSH data available.


Related in: MedlinePlus