Escherichia coli bl21 de3

The correctly sequenced EgAnxB3 and EgAnxB38 plasmids were digested with restriction enzymes, ligated into the pET32a(+) plasmid. The resulting recombinant plasmids were transformed into Escherichia coli BL21 (DE3) (Tiangen, Beijing). E. coli cells containing pET32a-EgAnxB3 and pET32a-EgAnxB38 were cultivated at 37 °C for 8 h. Then the transformants were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside. The recombinant proteins were purified using a Ni²⁺ affinity chromatography column (Bio-Rad, Hercules, CA).

the corresponding primer pairs listed in Supplementary Data 1. The genomic DNA was extracted from A549 cells and was used as the template for probe amplification using primer pairs PlrpF/PlrpR or FAM-F/PlrpR. The optimized coding sequence of E2F1 (NP_005216.1) was inserted into the plasmid pGEX-4T-1 at BamHI/XhoI sites to be expressed by fusion with GST in Escherichia coli BL21 (DE3, TIANGEN, China). The GST-E2F1 fusion protein was purified based on the protocol of GST 4FF Sefinose (TM) Resin Kit (Sangon Biotech, China). 20 ng FAM labeled probes were incubated with GST-E2F1 fusion protein in a 20-μl reaction mixture at 25 °C for 30 min. For competition assays, 0.4 μg of the unlabeled specific probe or the nonspecific probe sperm DNA was added to the binding reaction mixture. Then the samples were loaded on 4.5% (w/v) native polyacrylamide gels for electrophoresis. The fluorescently labeled DNA was detected by Amersham ImageQuant 800 (GE, America). The sequences of all probes and PCR primers we used in the EMSA assay were listed in Supplementary Data 1.

S. iniae SF1 (serotype I) is a pathogenic strain that had caused an epidemic in farmed flounder [21] (link). It was cultured in TSAYE medium [22] (link) at 28°C. Escherichia coli BL21(DE3) (used in this study as a host strain for expression and purification of recombinant proteins) was purchased from Tiangen (Beijing, China) and cultured in Luria-Bertani broth (LB) medium at 37°C.

The recombinant plasmids, pET‐28a (+)‐att, pET‐28a (+)‐trap, and pET‐28a (+)‐eAls, were transformed into in Escherichia coli BL21 (DE3) (Tiangen) and were expressed the ATT, TRAP, and eAls proteins with 0.1 mM isopropyl‐β‐D‐1‐thiogalactopyranoside (IPTG, Biosharp) induction at 37°C for 4 h, respectively. Then, the bacterial cells were obtained by centrifugation and were ultrasonicated, and the suspension was acquired. The His‐tagged ATT, TRAP, and eAls proteins were purified by using His‐binding‐resin (Novagen) according to the manufacturer's instructions. These proteins were confirmed with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) and Western blot. For Western blot, anti‐His tag monoclonal antibodies (mAbs) (Sigma) and horseradish peroxidase (HRP)‐conjugated goat antimouse IgG antibodies (Sigma) were used as the primary antibodies, the secondary antibodies, respectively.

The cDNA of chloropin (UniProt B3ECZ8) derived from Chlorobium limicola was synthesized by GENEWIZ (Nanjing, China). Escherichia coli BL21 (DE3) was purchased from Tiangen. Columns for protein purification were purchased from GE Healthcare. SDS-PAGE precast gels and MOPS Running Buffer were purchased from Genscript. Proteases, including human neutrophil elastase (HNE), pancreatic elastase, trypsin, kallikrein 1 (KLK1), kallikrein 7 (KLK7), proteinase 3 (PR3), thrombin, activated factor IX (FIXa), activated factor X (FXa), activated factor XI (FXIa), activated protein C (APC), tissue-type plasminogen activator (tPA), cathepsin B and cathepsin G, were purchased from Haematologic Technologies. The fluorescence-quenched KLK7 substrate Abz-Asn-Leu-Tyr-Arg-Val-Glu-Gln-Lys (Dnp) was synthesized from BankPeptide, while the thrombin substrate S-2238 was purchased from Chromogenix. Low molecular weight heparin (LMWH) with an average molecular weight of approximately 5 kDa was purchased from Sigma. All crystallization reagents and kits were purchased from Hampton Research.

The Escherichia coli BL21 (DE3) and Escherichia coli DH5α were purchased from Tiangen (Beijing, China). The pET-Amp plasmid was saved in our laboratory. Reference strains Staphylococcus aureus CVCC 1882 and Escherichia coli K88 were stored in our laboratory. Pseudomonas aeruginosa 337005 was purchased from BeNa Culture Collection (Beijing, China).

After identifying a full-length PTK cDNA from S. scabiei transcriptome data (GenBank: KY080515), the ORF encoding SsPTK was amplified. Primers (Invitrogen, Beijing, China) were designed using Primer 5.0 software and were as follows: forward, 5′-CGG GAT CCA TGC TCA AAA GTT ACG CTG TTC-3′, with a BamHI restriction site (underlined); reverse, 5′-CGA GCT CTT ATG TTA TCG TAG ATG TTG TTG CT-3′, with a SacI restriction site (underlined). SsPTK was amplified by PCR with cycling conditions of 94 °C for 5 min, followed by 35 cycles of amplification at 94 °C for 45 s, 62 °C for 45 s, and 72 °C for 45 s, and a final extension at 72 °C for 10 min. The resulting fragment was cloned into the pET32a(+) expression vector (Invitrogen, Beijing, China), and the construct was transformed into Escherichia coli BL21 (DE3) (TIANGEN, Beijing, China) for protein expression which was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) at 37 °C for 10 h. Recombinant SsPTK protein was purified from inclusion bodies in denaturing conditions (8 M urea) by chromatography using a Ni-NTA His-tag affinity kit (Bio-Rad, USA) according to the manufacturer’s instructions.