Hello everyone !
Do any people know why heterologous expressed protein break into two parts ? I do not know whether this happened during expression or purified, my target protein is ~90 kDa. So i have to use two tags to purified it, which can assure me get intact protein. As i will introduce it into mammalian cells, so i have to make sure it expressed in intact form.
Did there exist any methods can ensure it expressed intactly ?
Thanks!
This is probably due to the presence of a protease hypersusceptible site. Changing the expression system may solve the problem if the new expression system lacks the proteolytic activity. Another approach would be to identify the site of cleavage by N-terminal sequencing of the C-terminal fragment, then introduce a mutation to alter the hypersensitive site.
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