thermocellum and C. josui scaffolding proteins in this study. The C. thermocellum strain F1 was used for the isolation of genomic DNA (Sakka et al., 1989). Escherichia coli strains XL1-Blue and BL21(DE3) RIL (Novagen) were used for the cloning and expression of parts of the C. thermocellum http://www.selleckchem.com/products/NVP-AUY922.html xyn10C and xyn11A genes (DDBJ accession nos. D84188 and AB010958, respectively). Recombinant E. coli strains were cultured in Luria–Bertani (LB) broth supplemented with ampicillin (50 μg mL−1) or kanamycin (50 μg mL−1) and chloramphenicol

(25 μg mL−1) at 37 °C. The DNA region encoding the native Xyn11A dockerin was amplified by PCR from the plasmid pKS101-1 (Hayashi et al., 1999) using the primers XynADF and XynADR (Table 1), digested with EcoRI and SalI and inserted into the EcoRI and SalI sites of pBluescipt II KS(+). After checking its nucleotide sequence, the inserted DNA fragment was cleaved from the recombinant plasmid using EcoRI and SalI, and inserted www.selleckchem.com/products/Everolimus(RAD001).html into the expression vectors pGEX-4T-1 (GE Healthcare) and pMAL-c2 (New England Biolabs). pGEX-4T-1 yields protein fused with glutathione S-transferase (GST) and pMAL-c2

yields the E. coli maltose-binding protein (MBP) fusion. Mutations in the first and/or the second segments of the dockerins were introduced by an overlapping PCR technique using various primer combinations: primers XADmut1R and XADmut1F were used to introduce mutations into the first segment using pKS101-1 as the template and primers XADmut2R and XADmut2F were used to introduce mutations into the second segment. To produce proteins with mutations in both segments, a second round of mutations was introduced into those mutant dockerin genes already containing mutations in the first segment. A similar method was used to amplify the DNA region encoding both the native and the mutant Xyn10C dockerins from the plasmid pKS103 (Hayashi et al., 1997) using the primers listed in Table 1. These were then inserted into pGEX-4T-1. The amino acid sequences of the native and mutant dockerins are shown

in Fig. 2b and c. The recombinant cohesin proteins used in this study were rCoh1-Ct and rCoh3-Ct 3-oxoacyl-(acyl-carrier-protein) reductase derived from C. thermocellum CipA, and rCoh1-Cj and rCoh6-Cj derived from C. josui CipA. All the recombinant cohesin proteins were produced and purified as described previously (Jindou et al., 2004). Escherichia coli XL1-Blue, containing one of the pGEX-4T-1 derivatives, was grown at 37 °C in LB broth supplemented with ampicillin (50 μg mL−1). When the OD600 nm reached 0.6, isopropyl-β-d-thiogalactopyranoside was added to the culture to a final concentration of 1 mM. After incubation at 37 °C for 3 h, the cells were collected by centrifugation at 3000 g for 10 min, and suspended in 0.1 M phosphate-buffered saline (pH 7.2). The cells were disrupted by ultrasonication and cell debris was removed by centrifugation at 10 000 g for 10 min.