ROLE OF PAIRED BASIC RESIDUES IN THE EXPRESSION OF ACTIVE RECOMBINANTGALACTOSYLTRANSFERASES FROM THE BACTERIAL PATHOGEN NEISSERIA-MENINGITIDIS

The lgtB gene encoding a beta-1,4-galactosyltransferase gene and the l gtC gene encoding an alpha-1,4-galactosyltransferase from the bacteria l pathogen Neisseria meningitidis were cloned into an expression vecto r and overexpressed in Escherichia coli, Both genes expressed very wel l, but problems with C-terminal proteolysis were encountered with both proteins. The lgtC protein was initially isolated from extracts of re combinant E.coli as a truncated species that retained enzymatic activi ty, and was subsequently shown by mass spectrometry to be 19 residues shorter than the expected protein. A specific set of engineered C-term inal deletions was constructed to investigate their effect on the expr ession of lgtC, As many as 28 residues could be deleted with little ef fect on activity, and with the concomitant improvement of the overall expression up to fivefold over the full length protein. The lgtB prote in was also proteolysed in extracts of normal E.coli strains into enzy matically inactive fragments lacking 28 or 41 C-terminal residues. Thi s degradation could be prevented by expression in an ompT protease def icient strain of E.coli. The full length lgtB protein was not stable i n soluble protein extracts from all recombinant strains, however a sta ble enzyme preparation could be achieved,vith the membrane fraction fr om cells of the ompT deficient strain expressing lgtB, Specific deleti ons of lgtB were also constructed, and 15 residues could be removed wi thout loss of enzyme activity and also with the concomitant improvemen t 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 segmen ts of similar to 50 amino acids frequently contained paired basic resi dues. Engineering of these segments may therefore be required as a gen eral practice to produce these enzymes for use in the large stale chem i-enzymatic synthesis of carbohydrate-based therapeutics.