T&Q re gene construction & expression

Joseph C. Bagshaw jbagshaw at wpi.edu
Sat Aug 12 21:22:25 EST 2000


First I would like to thank all those who respoded to my request for
advice on gene construction.  There are enough good ideas out there to
fill a book. (Hmmm.)  Using a combination of your suggestions, I was able
to construct, clone and sequence the gene in about one week.

Now here's my next question.  My gene encodes a 72 aa peptide, and I put a
stop codon at the end.  I intend to express the gene in Pichia, and the
peptide is not modified.  I want the peptide to be secreted from the cell
with concomitant cleavage so that the first amino acid encoded in my
synthetic gene becomes the N-terminal aa.  In other words, the secreted
peptide is identical to the natural product.  I would appreciate any
advice on choice of vector and host strains, and any other advice on any
aspect of this project.  I have a colleague who is an expert in producing
proteins in Pichia, but only after someone gives him a strain with a
plasmid already in it.  Thus it is up to me to build the plasmid and get
it into the right host strain.  Any suggestions will be welcome.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.






More information about the Methods mailing list