TABLE 1.
Plasmids used in this study
| Plasmid | Phenotype or genotype; oligonucleotides for PCR amplification or site-directed mutagenesis | Source or reference |
|---|---|---|
| pET24b | Kmr; expression vector | Novagen |
| pET15b | Apr; expression vector | Novagen |
| pSJS1240 | Spr; E. coli ileX and argU | 33 |
| pAMJL73 | Apr; 2.385 kb of M. jannaschii genomic DNA bases 524549 to 526933 of L77117 in pUC18; carries complete ORF MJ0591 (α subunit of 20S proteasome) | ATCCa |
| pAMJKH57 | Apr; 2.036 kb of M. jannaschii genomic DNA bases 1182020 to 1184055 of L77117 in pUC18; carries complete ORF MJ1237 (β subunit of 20S proteasome) | ATCC |
| pAMJHW03 | Apr; 2.157 kb of M. jannaschii genomic DNA bases 1114515 to 1116671 of L77117 in pUC18; carries complete ORF MJ1176 (proteasome-activating nucleotidase) | ATCC |
| pMjα | Kmr; 795-bp NdeI-to-EcoRI fragment generated by PCR amplification using pAMJL73; carries complete MJ0591 ligated into pET24b; produces the α subunit of the 20S proteasome; 5′-ctgcccatatgcaaatggtacctcc-3′ and 5′-gcgccgaattcttattcttcttgtt-3′ (NdeI and EcoRI sites indicated) | This study |
| pMjβ(Δpro) | Kmr; 669-bp NdeI-to-EcoRI fragment generated by PCR amplification using pAMJKH57; carries MJ1237 truncated at the 5′ end by 15 bp ligated into pET24b; produces the mature β subunit with an N terminus corresponding to Thr7 of the protein deduced from MJ1237; 5′-ctgcccatatgacaacaaccgttgg-3′ and 5′-gccgagaattcttatttgctctttc-3′ (NdeI and EcoRI sites indicated) | This study |
| pMjPAN1 | Apr; 1.6-kb NdeI-to-EcoRV fragment of M. jannaschii genomic DNA ligated into the HincII site of pUC19; carries 84 bp upstream and 191 bp downstream of the complete MJ1176 | This study |
| pMjPAN2 | Kmr; 1.6-kb EcoRI-to-HindIII fragment of pMjPAN1 ligated into pET24b; produces PAN and PAN(Δ1–73) | This study |
| pMjPAN3 | Apr; 1,312-bp fragment generated by PCR amplification using pAMJHW03; carries MJ1176 ligated into pUC19 using HincII; 5′-ccgcccatatggtttttgaagaatt-3′ and 5′-gcggcgaattcttatctgtagagaa-3′ (NdeI and EcoRI sites indicated) | This study |
| pMjPAN5 | Apr; 1,302-bp NdeI-to-EcoRI fragment of pMjPAN3 ligated into pET15b; produces full-length PAN with His6 at the N terminus and PAN(Δ1–73) | This study |
| pMjPAN6 | Apr; site-directed mutation of pMjPAN3 which disrupts the internal Shine-Dalgarno site; 5′-gagcttgaacgtgaaaatttacagttaatg-3′ (altered bases indicated) | This study |
| pMjPAN7 | Kmr; 1,302-bp NdeI-to-EcoRI fragment of pMjPAN6; carries the complete MJ1176 with the disrupted internal Shine-Dalgarno site; produces PAN of 47 kDa | This study |
| pMjPAN8 | Kmr; 1,083-bp NdeI-to-EcoRI fragment generated by PCR amplification using pAMJHW03; carries MJ1176 with a 219-bp 5′ deletion ligated into pET24b; produces PAN(Δ1–73); 5′-tccatatgaaagaaaatgagattttaa-3′ and 5′-gcggcgaaattcttatctgtagagaa-3′ (NdeI and EcoRI sites indicated) | This study |
a
ATCC, American Type Culture Collection.