Role of paired basic residues in the expression of active recombinant galactosyltransferases from the bacterial pathogen Neisseria meningitidis.
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.
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
W W
WW
Wakarchuk
A
A
Cunningham
D C
DC
Watson
N M
NM
Young
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism
D017931:DNA Primers; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005700:Galactosyltransferases