Expressing a human protein in Coli

Rick Davis richard..davis at worldnet.att.net
Tue Aug 12 21:30:20 EST 1997


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

Regards,
Rick



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