TABLE 1.
Plasmids and fusion proteins
| Plasmida | Terminus encodedb
|
Primersc | Sequence at junctiond | |
|---|---|---|---|---|
| Amino | Carboxyl | |||
| pAG6 | Wild-type PBP 6 | Wild-type PBP 6 | ||
| pPJ5C | Wild-type PBP 5 | Wild-type PBP 5 | ||
| pPJ5D | aa 1-385 of PBP 5 | Anchorless PBP 5 | ||
| pPJ5/4 | aa 1-385 of PBP 5 | 17 C-terminal aa of PBP 4 | A, B, C, D | RPLVVLQEIPEGN|PLVRFESRLYKDIY |
| pPJ5/6 | aa 1-376 of PBP 5 | 31 C-terminal aa of PBP 6 | A, E, F, G | QLDGKTIEQRPLV|VMENVEEGGFFGRV |
| pPJ5/D | aa 1-376 of PBP 5 | 23 C-terminal aa of DacD | A, H, I, J | QLDGKTIEQRPLV|VTLESVGEGSMFSR |
| pPJ5/W | aa 1-385 of PBP 5 | 58 C-terminal aa of ProW | A, K, L, M | RPLVVLQEIPEGN|LRGIGRLDMGLATV |
| pPJ5I/6II | aa 1-294 of PBP 5 | 113 C-terminal aa of PBP 6 | A, N, O, G | KKLLTWGFRFFET|VTPIKPDATFVTQR |
| pPJ6I/5II | aa 1-287 of PBP 6 | 109 C-terminal aa of PBP 5 | P, Q, R, S | EKLLTWGFRFFET|VNPLKVGKEFASEP |
All plasmids were constructed by cloning DNA fragments into the NheI-HindIII sites of pBAD18-CAM (Cmr). Plasmid pAG6 replaces pPJ6 and was constructed according to the description for pPJ6 as described previously (19). Plasmids pPJ5C and pPJ5D were described previously (19).
The number of amino acids and source of the protein encoded by each cloned DNA fragment. Each plasmid expressed a single hybrid PBP consisting of the amino acid segments indicated. The amino acids are numbered from the initiating methionine of the complete open reading frame. Subtract 29 from this number to arrive at the residue position in the mature protein, which is the convention adopted for reporting residue locations in the crystal structure.
Oligonucleotide primers used to construct the two segments of each of the cloned DNA fragments. The sequence of each oligonucleotide is given in Table 2. The oligonucleotides are presented in the order P1, P2, P3, and P4, as described in Materials and Methods.
Partial amino acid sequence (one-letter code) just before and after the junction site (vertical line) of hybrid PBPs, as encoded by the plasmids listed. The underlined sequences in pPJ5I/6II and pPJ6I/5II represent the α 10 helix and the first half of the β12 β-barrel that define the junction between domains I and II in the crystal structure of PBP 5 (4).