anti-betagalactosidase antibodies & lacZ fusions

PHILLIPSA at LARS.AFRC.AC.UK PHILLIPSA at LARS.AFRC.AC.UK
Fri Feb 21 04:41:00 EST 1992


Vectors such as the pUC series and Bluescript contain
the lacZ-alpha peptide coding sequence (less than 200bp,
I think), the product of which complements the (much larger)
C-terminal polypeptide produced by the host. Antibodies to
B-galactosidase may recognize the alpha peptide, but the multiple
cloning site in these vectors is located in the coding sequence after
about the fifth codon. The cloning site itself is essentially
random sequence at the polypeptide level, being a convenient
series of restriction sites, with no relation to B-gal. An in-frame
insert in the cloning site will therefore produce a fusion protein
with just five amino acids of beta-gal, unless the insert size is a
multiple of three and has no in-frame stop codon, in which case
translation will continue through into the C-terminal part of the
lacZ-alpha. E.coli then probably removes the N-terminal Met, leaving
just four amino acids for the antibodies to recognize. This is
pushing your luck.
The reason that vectors like lambda-gt11 produce an immunogenic fusion
protein is that these have the whole of the B-gal gene in and the cloning
sites are at the 3' end of the coding sequence - so you have 100kD or
so of polypeptide for the antibodies to get their teeth on.

Andy Phillips
Long Ashton Research Stn
Bristol, UK




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