I am not much aware of pichia system but in general, its quite possible your
protein is getting protelysed and all that’s leftover is gfp. Although I expect
mbp to be there as well as its quite well behaved tag. You might want to
analyze whole cell lysate to see if your protein is there. A time point
analysis for expression might be helpful. You might want to express it for less
time.
Dhiraj
________________________________
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of dongxiaofei
<18811070...@163.com>
Sent: Friday, October 26, 2018 12:40:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Only C-terminal GFP-tag of fusion protein expression in
Pichia Pastoris.
Dear All,
I'm focusing on expressing my fusion protein in Pichia pastoris. The
size of my protein is about more than 200 kDa, and there are mbp-tag and
GFP-his-tag at N-terminus and C-terminus. Verify that my pPICZb plasmid is no
problem by sequencing.
After centrifugation, load the supernatant to amylose column and NI column,
only NI beads have green fluorescence. No strips in the eluent of amylose, and
a strip of gfp-tag for NI column in SDS-PAGE.
Only C-terminal GFP-tag of my fusion protien was expressed !
Why pichia pastoris expresses C-terminal GFP-tag, but neglect my protein
sequence? What should I do to solve this problem and get my protein in Pichia
Pastoris?
Would be very grateful for any advice!
Thanks,
Dong Xiao
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