| Autor: |
Jian Zhong Ma, Zhi Xin Di, Yong Gang Wang |
| Rok vydání: |
2014 |
| Předmět: |
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| Zdroj: |
Advanced Materials Research. :248-251 |
| ISSN: |
1662-8985 |
| DOI: |
10.4028/www.scientific.net/amr.998-999.248 |
| Popis: |
A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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