TABLE 1.
Bacterial strains, plasmids, and primers used for sequencing in the present study
Restriction enzyme sites are underlined. Apr, ampicillin-resistant; Kanr, kanamycin-resistant.
| Strains and plasmids | Description | Source |
|---|---|---|
| Strains | ||
| Vibrio sp. FC509 | A chondroitin sulfate-degrading marine bacterium that secretes multiple GAG lyases (patented as CGMCC 8913) | This study |
| E. coli BL21(DE3) | F−, ompT, hsdSB (rB−, mB−), dcm, gal,λ (DE3), pLysS, Cmr | Novagen |
| E. coli Top10 | F− mcrA Δ (mrr-hsdRMS-mcrBC) ϕ80lacZΔM15 ΔlacX74 deoR recA1 araD139Δ (araA-leu)7697 galU galK rpsL endA1 nupG | Invitrogen |
| Plasmids | ||
| pBAD/gIII A | Expression vector; Apr | Invitrogen |
| pCold TF | Expression vector; Apr | Takara |
| pET22b | Expression vector; Apr | Novagen |
| pET30a | Expression vector; Kanr | Novagen |
| pET22b-HCLase | pET22b carrying an amplified NcoI-XhoI fragment encoding the recombinant protein of HCLase fused with a His6 tag at the C terminus | This study |
| Sequencing primers | ||
| HCLase-F | 5′-GCCATGGATATGCGATGACCACCAGTTCACTG-3′ | |
| HCLase-R | 5′-GCTCGAGTCGCACTGAAAATTGATAACTTTGTCC-3′ | |
| HCLase-W492A-F | 5′-CCCGTGGTCGATAACCATCGACTCGC-3′ | |
| HCLase-W492A-R | 5′-TGCGTAATCGGTGTAGTGGTCTTGCTGGC-3′ | |
| HCLase-H285A-F | 5′-GACATTGCTTATAACGGCACTTATGGC-3′ | |
| HCLase-H285A-R | 5′-GCCTGCCTGAATAAAGGAGCCATCTTGG-3′ | |
| HCLase-Y290A-F | 5′-AACCGGCACTTATGGCAACGTGCTACTGGG-3′ | |
| HCLase-Y290A-R | 5′-TGCAGCAATGTCGCCATGCTGAATAAAGGAG-3′ | |
| HCLase-Y294A-F | 5′-GGCAACGTGCTACTGGGTGGGCTTGGC-3′ | |
| HCLase-Y294A-R | 5′-TGCAGTGCCGTTATAAGCAATGTCGCATGCTG-3′ | |