Maltose Binding Protein-Fusion Proteins

Humberto Oritz-Zuazaga zuazaga at ucunix.san.uc.edu
Fri Sep 13 23:23:30 EST 1991


I am posting this on behalf of my wife, who has no newsgroup access,
but will very soon, if I can help it :-). Replies to me will be
summarized if there is sufficient interest.

---cut here---
Hi!  I am trying to produce MBP-fusion proteins using the New England
BioLabs fusion-protein expression system.  I am using their vector
pIH902 which has an exact deletion of the malE signal sequence.  So
far, I have succesfully inserted my cDNAs (in frame) into the vector,
confirming it by diagnostic endonuclease digestions, PCR amplification
of inserts of correct size from the vectors, and sequencing of the
ligation ends.  However, when attempting to express the fusion
proteins, I find that the induced bands on PAGEs are 60% the expected
molecular weight.  I have not purified any of my fusions; my PAGEs have
been done with uninduced and induced cell extracts.

I have thought of many possibilities:  (a)  the fusion proteins are
running anomalously during PAGE;  (b)  the fusion proteins are being
degraded;  (c)  a stop codon has somehow been produced leading to an
incomplete protein; etc.

Has anyone worked with this system?  If so, could you give me your input
in terms of what could be happening?  I'd like to hear from anyone who
has experience with this system or any other similar one.

Sincerely,

Sandra Pena de Ortiz
Graduate Student, Toxicology Program
Dept. of Environmental Health
University of Cincinnati
-- 
Humberto Ortiz-Zuazaga                      INTERNET: zuazaga at ucunix.san.uc.edu
Dept. of Physiology & Biophysics              BITNET:              picone at ucbeh
University of Cincinnati                         CIS:                72301,2303



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