Protein Engineering, Vol 11, 295-302, Copyright © 1998 by Oxford University Press
WW Wakarchuk, A Cunningham, DC Watson and NM Young
The lgtB gene encoding a beta-1,4-galactosyltransferase gene and the lgtC
gene encoding an alpha-1,4-galactosyltransferase from the bacterial
pathogen Neisseria meningitidis were cloned into an expression vector and
overexpressed in Escherichia coli. Both genes expressed very well, but
problems with C-terminal proteolysis were encountered with both proteins.
The lgtC protein was initially isolated from extracts of recombinant E.coli
as a truncated species that retained enzymatic activity, and was
subsequently shown by mass spectrometry to be 19 residues shorter than the
expected protein. A specific set of engineered C-terminal deletions was
constructed to investigate their effect on the expression of lgtC. As many
as 28 residues could be deleted with little effect on activity, and with
the concomitant improvement of the overall expression up to fivefold over
the full length protein. The lgtB protein was also proteolysed in extracts
of normal E.coli strains into enzymatically inactive fragments lacking 28
or 41 C-terminal residues. This degradation could be prevented by
expression in an ompT protease deficient strain of E.coli. The full length
lgtB protein was not stable in soluble protein extracts from all
recombinant strains, however a stable enzyme preparation could be achieved
with the membrane fraction from cells of the ompT deficient strain
expressing lgtB. Specific deletions of lgtB were also constructed, and 15
residues could be removed without loss of enzyme activity and also with the
concomitant improvement of the overall expression up to twofold over the
full length protein. Longer deletions produced protein but activity could
not be detected in these recombinant strains. Examination of the
glycosyltransferase sequences from a wide range of bacteria showed their
C-terminal segments of approximately 50 amino acids frequently contained
paired basic residues. Engineering of these segments may therefore be
required as a general practice to produce these enzymes for use in the
large scale chemi-enzymatic synthesis of carbohydrate-based therapeutics.
ARTICLES
Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
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