[The analysis of heterogeneity of HWTX-I expressed in Pichia pastoris].
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.
Nie
Dong-Song
DS
Department of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414000, China.
Zhou
Yan-Kai
YK
Cao
Zuo-Ying
ZY
Liu
Yu
Y
D000818:Animals; D009498:Neurotoxins / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D030162:Reptilian Proteins / Q000096:biosynthesis* / Q000235:genetics; D013111:Spider Venoms / Q000096:biosynthesis* / Q000235:genetics
D009498:Neurotoxins; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I