A polyclonal rabbit antiserum generated toward the Pet passenger domain has been described previously (Eslava et al., 1998). Secondary goat anti-rabbit antibodies conjugated with alkaline phosphatase (AP) and AP-substrate (5-bromo-4-chloro-3-indolylphosphate) were obtained from Sigma-Aldrich (UK). DNA-modifying enzymes were purchased from New England Biolabs (UK) and used according to the manufacturer’s instructions. Bacteria were grown at 37 °C in Luria–Bertani

(LB) broth and where necessary, the growth medium was supplemented with 100 μg mL−1 ampicillin, 2%d-glucose or 0.02%l-arabinose. HEp-2 cells used for cytotoxicity Sirolimus cell line assays were propagated at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum gold (PAA Laboratories, Austria). The Escherichia coli strain used in this study was HB101 (Promega, UK). Plasmids used in this study are listed in Table 1. A codon-optimized

pet gene was synthesized de novo by GenScript and cloned into pBADHisA (Invitrogen, UK) to generate pBADPet. A 323 bp MluI–BglII fragment comprising the Pet signal peptide without the N1H1 (ESPR) region was synthesized de novo and cloned into pUC57 (GenScript). pUC57ΔN1H1 was digested with MluI and BglII and subcloned into pBADPet, predigested with the same restriction enzymes, to generate pBADPetΔN1H1. To construct the chimeric signal sequence (ss)-pet constructs, the NcoI restriction Olaparib cost site within the pet ORF in pCEFN1 (Eslava et al., 1998) was altered through site-directed mutagenesis using the QuickChange II kit (Stratagene) and the oligonucleotides 5′-ACTTGGAACAACCCACGGAATAATAGG-3′ (Pet1Fw) and 5′-CCTATTATTCCGTGGGTTGTTCCAAGT-3′ (Pet1Rv). The resulting construct, pCEFN1(NcoI), was amplified by PCR using oligonucleotides 5′-AAAAACCATGGATATATCTAAAGCATGGGCC-3′ (Pet2Fw) and 5′-GCAACTCTCTCAGGGCCAG-3′ (Pet2Rv) to generate a DNA fragment encoding Pet lacking its signal peptide (Met55–Phe1295). The resulting amplicon and the target vectors containing

signal sequences from the genes malE, dsbA and phoA, pCFS117 (pTRC99a+malEss), pCFS119 (pTRC99a+dsbAss) and pCFS122 (pTRC99a+phoAss) (Schierle et al., 2003) were then digested with NcoI and KpnI and ligated to generate the chimeric ss-pet constructs, pMBPssPet, pDsbAssPet ID-8 and pPhoAssPet. The control construct, pPetssPet, was generated through the removal of trxA from construct pPetssTrxA (Desvaux et al., 2007) by inverse PCR using oligonucleotides 5′-AAAAGGTACCAGCTTGGCTGTTTTGGCGG-3′ (Pet3Fw) and Pet1Rv, digestion with NcoI and KpnI and ligation with pet amplified from pCEFN1(NcoI) predigested with the same restriction enzymes. Overnight E. coli HB101 LB cultures, supplemented with glucose and transformed with pBADPet or pBADPetΔN1H1, were diluted 1 : 100 into a fresh medium and grown to an OD600 nm=0.5.