Development of the production process for double-tagged fusion protein
| Autor: | Pei-Jie Li, 李佩潔 |
|---|---|
| Rok vydání: | 2011 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 99 The conventional affinity-tag fusion technology has provided a simple and convenient method for the purification of recombinant proteins through genetic engineering. However, affinity tag removal is generally difficult and expansive for large-scale purification of target protein. In order to avoid using costly protease and simplify purification process, the self-cleaving fusion-tag has recently been combined with additional purification tag to generate novel and convenient protein purification methods. In this work, a self-cleaving purification tag (e.g. pH-induced intein, Ssp DanB, New England Biolabs) was introduced between GST (glutathione-S-transferase) and EGFP-His (enhanced green fluorescent protein). The recombinant protein expression plasmid, pGIE-30, encoding the GST-intein-EFFP (GIE) fusion protein was chosen as a model system. Initially, it was to find the optimal environmental conditions for the production of GST-intein-EGFP fusion protein. The operating conditions was initial culture pH of 7.0, the volume of inoculums of 1.0%, the induction temperature of 25℃, and the culture time of 12 hr. The optimal conditions were then employed to scale up the GST-intein-EGFP production in a 5 L bioreactor. Subsequently, the collected cells were suspended with buffer (pH 8.0) and disrupted by ultrasound cell disrupter to release the soluble fusion protein at 20 kHz and 4℃. The release of soluble protein, GST activity and EGFP intensity from 2% (ww/v) disrupted cells was found to be 2.31 (mg/ml), 4.69 (U/ml) and 1.01×105 (AU/ml), respectively. The fusion protein was more stable at around pH 7-9 and lower temperature (4℃). To design the purification process, the double-tagged fusion protein with both GST and His tags would be specially adsorbed by GSTrap and HisTrap affinity columns, respectively (GE Healthcare) at pH 7.5 from clarified E. coli feedstock in small packed beds (1 ml). For GSTrap column, the adsorbed target protein (EGFP) can be released from the adsorbent by intein self-cleavage occurring through a pH change from 7.5 to 6.0 at 4oC. For HisTrap column, the GST can be firstly removed from the adsorbent by intein self-cleavage occurring at pH 6.0 and 4oC. The adsorbed EGFP can be further eluted by using 300 mM imidarzole. The results showed that the HisTrap column was found to be more effective for the recovery of EGFP as compared to the GSTrap column. Finally, the immobilized (0.1 M Ni2+) STREAMLINE chelating adsorbent (10 ml) (GE Healthcare) was employed to evaluate the purification efficiency for the target EGFP in packed beds (i.d. 1.6 cm). At first elution step with pH 6 buffer, the GST activity was released with a yield of 41.06%. At second elution step with 300 mM immidarzole, the EGFP intensity was recovered with a yield of 91.71% and a purification factor of 2.82. The SDS/PAGE of partially purified GST and Intein-EGFP-His at these elution steps yields two bands with molecular weights of 25 kDa and 53 kDa, respectively. The results showed that the intein-mediated protein purification process could be employed for the production of EGFP at a large scale. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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