TABLE 1.
Plasmids used
| Plasmid | Propertiesa | Source or reference |
|---|---|---|
| pSKII− | Ampr, high-copy-number expression vector, T7 promoter | Stratagene |
| pSKE8 | pSKII+, carrying lacS of S. thermophilus A147 as 4,073-bp EcoRI fragment (galM+ lacS+) | 19 |
| pSKE8E | pSKE8 with additional EcoRI site 21 bp upstream of the initiation codon of lacS | 9 |
| pSKE8his | pSKE8 with His-tagged lacS | 9 |
| pSKE8N | pSKE8 with NcoI site at the initiation codon of lacS | 9 |
| pSKE8(lacS-H552R) | pSKE8 with His-552 of LacS replaced by Arg | 19 |
| pSKE8(lacS-I548E/G556D) | pSKE8 with Ile-548 and Gly-556 of LacS replaced by Glu and Asp, respectively | 19 |
| pSKoppAChis | pSKE8his with lacS (1,936-bp NcoI-MluI fragment) replaced by oppA | 16a |
| pKK223-3 | Ampr, medium-copy-number expression vector, taq promoter | Pharmacia |
| pSK181 | pSKII−, carrying a 543 bp fragmentb that specifies the C-terminal 181 amino acids of LacS | This work |
| pSK173 | pSKII−, carrying a 519-bp fragmentb that specifies the C-terminal 173 amino acids of LacS | This work |
| pSK154 | pSKII−, carrying a 462-bp fragmentb that specifies the C-terminal 154 amino acids of LacS | This work |
| pKKELE | pKK223-3, carrying a 631-bp fragmentb that specifies IIALacS | This work |
| pSKIIAhis | pSKE8N with the 2,243-bp NcoI-BamHI fragment replaced by a 644-bp NcoI-BamHI fragment that specifies IIALacS-6H | This work |
| pSKIIAm1his | pSKIIAhis with H552R | This work |
| pSKIIAm2his | pSKIIAhis with I548E/G556D | This work |
| pGKHis | pGK13, carrying His-Tagged lacS of S. thermophilus as a 3,824-bp EcoRI-DraI fragment from pSKE8 ligated into the EcoRI-EcoRV sites | 9 |
a
Amp and Cm indicate resistance to ampicillin and chloramphenicol, respectively.
b
PCR fragment blunt-end ligated in the SmaI site of the vector.