The analysis of heterogeneity of HWTX-I expressed in Pichia pastoris.
- Author:
Dong-Song NIE
1
;
Yan-Kai ZHOU
;
Zuo-Ying CAO
;
Yu LIU
Author Information
1. Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414000, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Neurotoxins;
biosynthesis;
genetics;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
Reptilian Proteins;
biosynthesis;
genetics;
Spider Venoms;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2006;22(2):215-219
- CountryChina
- Language:Chinese
-
Abstract:
To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.
