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Advances in Applied Biotechnology Part 9

Tham khảo tài liệu 'advances in applied biotechnology part 9', khoa học tự nhiên, công nghệ sinh học phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | Improvement of Heterologous Protein Secretion by Bacillus subtilis 149 We showed that the secretion production and activity of hIFN-a2b with propeptide increased by more than 3-fold compared to that without propeptide. The amount of secreted hIFN-a2b with propeptide was 15mg L. This result indicated that the propeptide of AmyE enhanced the secretion of hIFNa-2b Fig. 3 Kakeshita et al. 2011a . IP hlFN-p SP-AmyE SP-Pro-AmyE Pro-IFN-p IFN-P Fig. 4. Western blot analysis of hIFN-P production by B. subtilis Dpr8 with pHKK3111 AmyE SP-hIFN-P or pHKK3211 AmyE SP-Pro hIFN-P . Samples were collected at 20 h after xylose induction separated by 15 SDS-PAGE and stained with Western blotting using anti hIFN-P polyclonal antibodies. Dpr8 with pHKK3111 lanes 1 and 2 Dpr8 with pHKK3211 lanes 3 and 4 0.6 xylose induced lanes 1 and 3 none induced lanes 2 and 4 and commercially purified hIFN-P 50 ng lane 5 . Arrowheads indicate the positions of the Pro-hIFN-P and hIFN-P. adapted from Kakeshita et al. 2011b In L. lactis directed mutagenesis experiments demonstrated that the positive effect of LEISSTCDA on protein secretion was due to the insertion of negatively charged residues in the N-terminus of the mature moiety Le Loir et al. 2001 . In hIFN-a2b with AmyE propeptide the first 10 amino acid residues of this mature protein have a net charge of -1. On the other hand hIFN-a2b without propeptide has a net charge of 0. In addition we demonstrated that propeptide mutants of neutral or positive charge resulted in a reduction in the amount of secreted hIFN-a2b compared with propeptides of negative charge. This result suggested that negative charges in the mature protein can enhance the secretion of hIFN-a2b Kakeshita et al. 2011a . We then indicated that the AmyE propeptide enhanced the secretion of the hIFN-P protein from B. subtilis as well. The secretion production and activity of hIFN-P with propeptide increased by more than 4-fold Fig. 4 Kakeshita et al. 2011b . The amount of secreted