In this study, we used E. coli strain BL21 (DE3) to express the recombinant protein his-tag streptavidin. To find out the optimal production conditions, we studied on the culture conditions, medium composition, induction conditions and the timing of induction. In the purification processes we tried to find out the difference between hydrophobic column and affinity column. We also tested the effect of heat treatment on the crude extract to increase the recombinant protein yield. The results showed that when cultured in LB medium, the optimal culture conditions of recombinant protein expression are 37¢XC, pH 6.0 to 7.0, and the induction temperature is 37¢XC. The best induction time is at late log phase or the early stationary phase when OD600 values reached to the ranged of 1.1 to 1.8. The inducer, IPTG concentration is 0.1 mM, which can also replaced with 2 mM lactose. The best production medium is TB medium. When cultured in 5 liters fermentor with optimal culture and induction condition, the highest recombinant protein yield could be 81.1 mg /L. To improve the purification process, we used a affinity chromatography. The purified high homogeneous recombinant protein had a high biotin binding activity up to 14 U / mg, and the recovery yield could be as high as 97% in comparing with the hydrophobic column was only 51%. When we treated the crude extract with 75 ¢J for 10 min, the biotin binding activity was 14.1 U / mg, but the recovery rate decreased to 64 %.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0214108-144012 |
| Date | 14 February 2008 |
| Creators | Huang, Chi-tien |
| Contributors | I-Chen Hu, Jong-Kang Liu, Chan-Shing Lin |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0214108-144012 |
| Rights | off_campus_withheld, Copyright information available at source archive |
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