Pet24a

E. coli BL21 (DE3), E. coli DH5α, pET-24a (+) were provided by Invitrogen (CA, USA). pET-24a (+)-TmSm34, pET-24a(+)-TmSm48, and pET-24a(+)-TmSm84 were previously constructed by our laboratory. MTT were supplied by Solarbio (Beijing, China). Hoechst 33342 were purchased from Yeasen (Shanghai, China). All other reagents were of analytical grade and acquired from Sigma-Aldrich (Shanghai, China). Human lung cancer cell A549 and normal liver cell L-02 was obtained from the Type Culture Collection Committee of Chinese Academy of Science (Shanghai, China). Fetal bovine serum (FBS), penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin), Roswell Park Memorial Institute 1640 medium (RPMI1640), Dulbecco’s Modified Eagle medium (DMEM), DMEM/Nutrient Mixture F-12 medium (DMEM/F12), and B27 were purchased from Gibco (Waltham, USA). Basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were acquired from Pepcech (Cathy, USA).

E. coli host strains BL21, BL21 (DE3)pLysS and BL 21 Star^TM (DE3)plysS (Invitrogen) were used for gene expression experiments. Host strain DH5α (Invitrogen) was used as both expression and cloning strains. Vectors pET-24a and pET-24d (Invitrogen) were used for cloning and expression studies.
The Y. enterocolitica strain was isolated from the stool of a human patient. The protocol was according to Bhaduri and Wesley [27 (link)] with some modification. Fresh faecal sample was collected from the local hospital. The interval from sample collection to sample analysis in the laboratory was between 48 and 72 h. One gram from faecal sample was suspended in 9.0 mL of 0.1% peptone water and mixed in a blender for 30 s. One millilitre of the suspension was added to 9.0 mL of irgasan–ticarcillin–potassium chlorate broth in a tube and vortexed. The enrichment was held at room temperature for 48 h. Selectively enriched sample was vortexed and diluted 1:10 in 0.1% peptone water, and a 100 μL aliquot was plated on cefsulodin–irgasan–novobiocin agar and incubated at 30 °C for 24 h. Y. enterocolitica colony with a deep red centre was isolated. The identification of this strain was through the analysis of 16s rRNA gene sequencing.

The cDNA library of grouper fishes was prepared according to previous studies (Hsu et al, 2013 (link)). The -cDNA encoding EcHSP60 was amplified and cloned into the pET24a (Invitrogen) vector between the restriction enzyme cutting sites, NdeI and XhoI. The constructed plasmid was named pET-EcHSP60 and was used to express the recombinant EcHSP60 fused with a C-terminal six-His tag. To express the C-terminal truncated EcHSP60 removing the flexible GGM tail, the DNA region encoding E527 to G552 was removed from pET-EcHSP60 to form pET-EcHsp60^ΔC using In-Fusion seamless cloning kit (Takara Bio). In addition, the plasmids used to express the R264A, K266A, and R264AK266A mutants were also constructed by using the In-Fusion seamless cloning kit based on pET-EcHSP60 as the template.