Advice needed to subclone big inserts (6 kb) into pET system - (Feb/27/2013 )

Good day,

I need some advices from the experts here.

I need to subclone an amylopullulanase (~6 kb in size) into an expression vector, preferably pET system with His tag at both termini.

Any suggestion on the expression vector? Vectors other than pET systems are also welcome.

However, pET systems are normally >5 kb, so pET+insert would be >10 kb and E. coli may produce inclusion bodies.

Any suggestion on the choice of host?


Thanks in advance for the advices.

I think you need to do electroporation. 10 k is almost on the borderline for chemical competent cells.

It can be done in pET vector, I found this paper-

http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.0060163