Pqe30xa

Amino acids 40–374 of B. pertussis TcfA (Tohama I, UniprotKB Q79GX8) were cloned into pQE-30-Xa (Qiagen), which contains an N-terminal 6XHis-tag followed by a Factor Xa cleavage site. rTcfA-His was purified over HisPur Cobalt columns (Pierce). To generate tag-free rTcfA, purified rTcfA-His was treated with Factor Xa (New England Biolabs), then 1,5-Dansyl-Glu-Gly-Arg-chloromethyl (EMD Millipore), and passed again over HisPur Cobalt columns. Endogenous B. pertussis adenylate cyclase antigen and filamentous hemagglutinin were purchased from List Biological Laboratories (Campbell, CA).

cDNA coding for hr-anxA1 was amplified by polymerase chain reaction (PCR) using primers: 5’-GGTATCGAGGGAAGGGCAATGGTATCAGAATTC-3’ and 5’-GCTCAGCTAATTAAGCTTTAGTTTCCTCCACAAAGAGC-3’. The primers introduced Stu-I and Hind-III restriction sites, required for the ligation into the expression vector pQE30Xa (Qiagen). His-tagged hr-anxA1 was produced according to previously published protocol for anxA5 [25 (link)]. In short, E. Coli (SG13009 pREP4) (Novagen) were fermented in Luria-Bertani broth medium supplemented with Ampicillin (50ug/ml, Roche), Kanamycin (30ug/ml, Gibco) and 0.5% glycerol. At OD₄₅₀ of >6, over-expression of the protein was initiated by addition of 5mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Eurogentec). Proteins were purified by IMAC. Purity and homogeneity were assessed by silver-stained SDS-PAGE, western blotting and MALDI-TOF/TOF analysis.

Once the complete sequence of the halI gene was obtained, primers were designed to amplify the halI open reading frame (ORF) by PCR using the bacterial genomic DNA as a template and TaKaRa Ex Taq polymerase (see Table 2). Primers generated BamHI and SacI sites at the 5' and 3' ends of the amplicons, respectively (see Table 2). The DNA amplicon, 660 bp, containing the halI structural gene was digested with BamHI and SacI and ligated into vector pQE-30Xa (Qiagen), previously cut with the same restriction enzymes. This plasmid harboring the ORF of halI inserted downstream of the T5 promoter was named pQE-30Xa-halI068. Plasmid pQE-30Xa-halI068 was transformed into the expression strain E. coli XL1-Blue.
For overproduction of HalI, E. coli XL1-Blue cells carrying pQE-30Xa-halI068 were grown in dYT medium (tryptone 1.6%, yeast extract 1.0%, NaCl 0.5%, and glucose 0.2%) containing ampicillin (100 μg mL⁻¹) at 37°C under vigorous shaking. At an optical density at 600 nm of 0.5, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM. After 5 h incubation at 37°C, the cells were collected by centrifugation at 10,000 x g for 30 min and resuspended in 50 mM Tris-HCl (pH 8.0). Then, 3 μL of cell suspension were loaded onto SDS-PAGE (15%) in order to detect HalI overexpression.

5′ primers for cloning into the pET15b (Merck Millipore, UK) or pQE30-Xa (Qiagen, UK) plasmid were designed to anneal immediately after the signal peptide coding region, as predicted by the Signal P 3.0 server (http://www.cbs.dtu.dk/services/SignalP/), and 3′ primers were designed to include the stop codon of the gene (see Table S2 in the supplemental material). The cloning procedure was performed as outlined above for pALC2073, and ligated constructs were transformed into E. coli DH5α or XL1-Blue (for pQE30-Xa constructs) cells. pET constructs were isolated from DH5α cells using the QIAprep spin miniprep kit (Qiagen, UK) and transformed into E. coli BL21(DE3) cells. BL21 or XL1-Blue cells containing expression constructs were cultured in Luria broth containing 50 μg/ml ampicillin (Sigma-Aldrich, Dorset, UK) and induced in the mid-exponential phase of growth (OD₆₀₀ = 0.6) with 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) (ForMedium Ltd., Norfolk, UK) for 4 h. Cells were recovered by centrifugation at 8,000 × g and disrupted by using a French press, and His-tagged recombinant proteins were purified by affinity chromatography on a Ni-nitrilotriacetic acid (NTA) nickel affinity column (GE Healthcare, UK). Proteins were dialyzed by using Spectra/Por Float-A-Lyzer tubing with an 8,000 to 10,000 MWCO (Spectrum Laboratories, CA, USA).

In‐frame deletion mutants were constructed using the suicide vector pK18mobsacB by homologous double‐crossover recombination according to previous studies (Burckstummer et al., 2006; Wang et al., 2016). The insertional inactivation mutants used the suicide vectors pK18mob by homologous single‐crossover methodology (Qian et al., 2008; Kang et al., 2015). His₆‐tagged proteins were expressed using pET30a (Novagen) vectors and pQE30Xa (Qiagen) according to the manufacturer's instructions. To construct genetic complementation strains, the broad‐host vector pHM1 with inserts of full‐length sequences of genes of interest (under the control of the lacZ promoter) were established and electroporated into Xcc competent cells. The primers used to amplify the sequences are listed in Table S2.

cgt-BS gene from a previous study [36 (link)] was mutated with codon usage optimization. Escherichia coli JM109 [endA1, recA1, gyrA96, thi, hsdR17 (rk-,mk+), relA1, supE44, D (lac-proAB), F’ (tra D36, pro AB, lacIqZ Δ M15)] from Promega (Madison, WI, USA) was used as a host strain and plasmid pQE30xa (QIAGEN, Hilden, Germany) was used as an expression vector.