pPpiDΔParv was constructed as follows: a second EcoRV site was introduced at nucleotides

1062-1068 of ppiD by QuikChange mutagenesis of pPpiD using primers 5′-GTCTGGACGATATCCAGCCAGCGAAAG-3′ BYL719 ic50 and 5′-CTTTCGCTGGCTGGATATCGTCCAGAC-3′. In the resulting plasmid, the parvulin domain encoding sequence of ppiD was flanked by EcoRV sites. Deletion of the EcoRV fragment resulted in pPpiDΔParv. Plasmid pPpiDfs601 was made by cleavage of pPpiD with KpnI, removal of the resulting 3′-overhangs with DNA polymerase I Klenow fragment, and subsequent ligation. Plasmid pASKssPpiD for the production of a soluble periplasmic N-terminally hexa-His-tagged PpiD protein was constructed in three steps. First, a BamHI site was introduced at codons 33-34 of ppiD by QuikChange mutagenesis of pPpiD using primers 5′-GCGTGAGTGGATCCCTGATTGGCGGA-3′ and 5′-TCCGCCAATCAGGGATCCACTCACGC-3′. Second, the BamHI/HindIII fragment of the resulting plasmid, encoding PpiD without the transmembrane segment, MM-102 in vitro was cloned into the BamHI/HindIII sites of a pASKSurA plasmid that carried a SacI site at codons 22-23 of surA [2]. Third, the 5′-phosphorylated oligonucleotides 5′-CCATCACCATCACCATCACG-3′ and 5′-GATCCGTGATGGTGATGGTGATGGAGCT-3′ were annealed and cloned into SacI/BamHI of the above intermediate, thereby placing a

hexa-His sequence between the signal peptide sequence of surA and codons 34 to 623 of ppiD. To make pASKssPpiDΔParv, the SphI/PstI fragment of pASKssPpiD bearing the parvulin domain encoding sequence was replaced by a SphI/PstI fragment derived from plasmid pPpiDΔParv. To make pPpiDΔTM, a 1350 bp-fragment carrying the surA signal sequence-his 6 -ppiD fusion was PCR amplified from pASKssPpiD using primers 5′-CATTGATAGAGTTACGTAACCACTCCC-3′ and 5′-CACTTTCTGCTGCAGCGCG-3′. The product was cleaved with

SnaBI/PstI and cloned into the StuI and PstI sites of pPpiD. To create plasmid pSkp, a 1722 bp XhoI/NdeI fragment derived from plasmid pMP1 was cloned into the corresponding sites of pQE60 thereby removing the plasmid-encoded P T5 /O lac promoter/operator sequences. All plasmid sequences were confirmed by DNA sequencing. Table 3 Plasmids used in this study Plasmid Genotype Source, reference Thiamet G pACLacI pACYC184 derivative with lacI q ; CmR This study selleck screening library pASK75 vector, P/O tet , tetR, ColEI ori; ApR [60] pASKSurAa surA gene in pASK75; ApR [2] pASKSurAN-Ctb surAN-Ct fusion from pSurAN-Ct [2] in pASK75; ApR This study pASKssPpiD surA signal sequence-his6-ppiD (codons 34-623) fusion in pASK75; ApR This study pASKssPpiDΔParv pASKssPpiDΔ252-355; ApR This study pΩSurA Ω::spec-P Llac-O1 surA in pUC18; ApR; SpecR This study pMP1 skp gene region of E. coli MC1061 (corresponding to nucleotides 199495-201937 of the E. coli MG1655 genomec) in pSU18; CmR Gross laboratory pPLT13 mini-F carrying lacI q ; KanR [61] pPpiD ppiD gene and promoter of E. coli MC1061 (corresponding to nucleotides 460852-463020 of the E.