Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli
Background: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this re...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2015-01-01
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| Series: | Advanced Biomedical Research |
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| Online Access: | http://www.advbiores.net/article.asp?issn=2277-9175;year=2015;volume=4;issue=1;spage=66;epage=66;aulast=Khodadadi |
| _version_ | 1818555324610641920 |
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| author | Emad Khodadadi Mojtaba Panjepour Mahdi Abbasian Zahra Khalili Broujeni Mohammad Reza Mofid |
| author_facet | Emad Khodadadi Mojtaba Panjepour Mahdi Abbasian Zahra Khalili Broujeni Mohammad Reza Mofid |
| author_sort | Emad Khodadadi |
| collection | DOAJ |
| description | Background: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host.
Materials and Methods: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Results: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis.
Conclusion: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein. |
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| issn | 2277-9175 2277-9175 |
| language | English |
| last_indexed | 2024-12-12T09:51:52Z |
| publishDate | 2015-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
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| spelling | doaj.art-5897988421804dce80ab35ed303a85a72022-12-22T00:28:15ZengWolters Kluwer Medknow PublicationsAdvanced Biomedical Research2277-91752277-91752015-01-0141666610.4103/2277-9175.153886Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coliEmad KhodadadiMojtaba PanjepourMahdi AbbasianZahra Khalili BroujeniMohammad Reza MofidBackground: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. Materials and Methods: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. Conclusion: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein.http://www.advbiores.net/article.asp?issn=2277-9175;year=2015;volume=4;issue=1;spage=66;epage=66;aulast=KhodadadiCancerinsulin-like growth factor 1insulin-like growth factor 1 receptorInsulin-like growth factor binding proteins |
| spellingShingle | Emad Khodadadi Mojtaba Panjepour Mahdi Abbasian Zahra Khalili Broujeni Mohammad Reza Mofid Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli Advanced Biomedical Research Cancer insulin-like growth factor 1 insulin-like growth factor 1 receptor Insulin-like growth factor binding proteins |
| title | Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli |
| title_full | Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli |
| title_fullStr | Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli |
| title_full_unstemmed | Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli |
| title_short | Cloning and expression of full-length human insulin-like growth factor binding protein 3 (IGFBP3) in the Escherichia coli |
| title_sort | cloning and expression of full length human insulin like growth factor binding protein 3 igfbp3 in the escherichia coli |
| topic | Cancer insulin-like growth factor 1 insulin-like growth factor 1 receptor Insulin-like growth factor binding proteins |
| url | http://www.advbiores.net/article.asp?issn=2277-9175;year=2015;volume=4;issue=1;spage=66;epage=66;aulast=Khodadadi |
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