Protein Engineering Protocols - Mycobacteriology research center

290 Hecky et al.Fig. 4. Structure of wt TEM-1 β-lactamase (PDB identifier: 1btl). Residues frequentlymutated are shown in full atoms (e.g., M182T), the catalytic serine residue(S70) and the N- and C-terminus are indicated. Residues are numbered in agreementwith Ambler (38). Most mutated amino acid residues are located near to the protein surface.The figure was drawn using SWISS-PDB Viewer (31).in individual optimized variants were always scattered throughout the entirestructure and were never clustered adjacent to the site of deletion.3.7. Construction of Full-Length MutantsOptimized clone N∆5-S3/6 was re-elongated to elucidate to what extent the setof mutations compensating for protein destabilization after terminal truncationaffect the complete enzyme. The full-length clone named FL-S3/6 was constructedby adding the missing residues according to the schematic representationshown in Fig. 2A. This variant was designed for translocation to the periplasmand was expressed in Escherichia coli XL-1 Blue cells to probe its functionalityin vivo. Unexpectedly, the numbers of colonies on plates (20 h at 37°C) containingchloramphenicol and 100 µg/mL ampicillin was only 50% of the number of