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Correct protein expression in pichia??

pichia protein expression size

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#1 helpless

helpless

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Posted 31 January 2012 - 10:30 PM

Hi, all. I am new in this field and is facing some problems with the protein expression in PIchia pastoris system. I already cloned my gene into pPICZB for intracellular expression of the protein. Integration of the gene was confirmed by PCR and sequencing. Protein expression was carried out as suggested by the kit manual and protein expression was analyzed by SDS-PAGE and western blot. In SDS-PAGE, I saw protein overexpression in one band of more than 50 kDa (my protein is ~54 kDa) which correspond just correctly to my target protein. However, western blot with his tag antibody showed signal more than one band, the highest reactivity is with a band of more than 60 kDa. Apart from that, I tried to purify the protein and SDS-PAGE of the purified proteins showed single protein band of ~70 kDa. I'm quite worry whether is the protein is really my target protein. Could someone advice me? Thanks.





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