Expressing a human protein in Coli

bipin k. dalmia dalmiabk at
Wed Aug 13 11:28:47 EST 1997

On Wed, 13 Aug 1997 02:30:20 GMT, richard..davis at
(Rick Davis) wrote:

>Our lab has an interest in expressing a human tyrosinase protein in E.
>coli. This protein has a signal sequence which is not present in the
>mature peptide. In terms of designing a set of PCR primers for pulling
>this gene out of a cell line via RT-PCR and expressing in E. coli,
>would we be better off designing primers that ignore the signal
>sequence altogether (i.e. design a 5' primer homologous to the first
>amino acid of the mature form of the protein)? Or could we include the
>signal sequence in the hopes that the protein would fold properly in 
>E. coli even with the additional signal sequence residues at the amino
>terminus? We use the pET series of vectors in our lab routinely and
>our hope is to be able to express the tyrosinase intracellularly with
>either pET11a  or pET15b. Anyone out there worked on a project
>similar to this? TIA

i would recommend expressing without the native signal sequence. E.
coli would not be able to process the signal sequence and the added
hydrophobicity would lead to increased probability of inclusion body
formation (as if that isn't a big problem with e. coli already). if
you only want antibodies made then i guess inclusion bodies would be
okay but if you are looking at getting functional protein and the
mature protein expressed intracellularly is not, then you may try
using a bacterial signal sequence to target the protein to the
periplasm. pET22b (and a couple others) have signal sequences which
sometimes work.


Bipin K. Dalmia, Ph.D.
Protein Expression and Purification
Pioneer Hi-Bred International, Inc.
Johnston, Iowa 50131

dalmiabk at

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