His bind purification kit

The OXA enzymes extracted from successfully cloned carbapenemase-producing strains were purified using the His·Bind Purification Kit (Millipore, Burlington, MA, USA). Meropenem hydrolysis activity was directly detected using the BioTek Synergy NEO system, which measures the change in absorption light (Abs) at the wavelength indicated for meropenem (310 nm). Hydrolysis rates were calculated by comparing the change in optical density (OD) using Microsoft Excel. Additionally, an in vitro inhibitory test was performed to demonstrate the hydrolysis ability of the new OXA-type carbapenemase.

The LukS-PV sequence was amplified from PVL-positive Staphylococcus aureus isolates by polymerase chain reaction (PCR) using the following primers: LukS-FW, 5ʹ-acgcGGATCCGAATCTAAAGCTGATAACAATATTGAGAATATTG-3ʹ; LukS-RV, 5ʹ-accgCTCGAGTCAATTATGTCCTTTCACTTTAATTTCATGAG-3ʹ. Recombinant LukS-PV was generated as described previously [27 (link)]. Purification of recombinant LukS-PV was performed with the His-Bind® Purification Kit (Millipore, USA) in accordance with the manufacturer's instructions.

A plasmid containing human CXCL16-I123A181 cDNA with a C terminal flag and His-tag was purchased from Vigene Biosciences (Jinan, Shandong Province, China). Plasmids encoding CXCL16-I123V181, T123A181, and T123V181 cDNA mutations were generated by site-directed mutagenesis (Stratagene, La Jolla, CA, United States). The primers for 123T were 5′-TGAGGCCTGAGAAgTTGGGGGGCTGGTAGGAA-3′ (forward) and 5′-CAACTTCTCAGGCCTCAGAGGGGGCA-3′ (reverse). The primers for 181V were 5′-CCCAaCTGCC AGACTGTGGCCCGCA-3′ (forward) and 5′-ACAGTCTG GCAGtTGGGCCTGAGGCTGGGGA-3′ (reverse). The sequences of the four plasmids were checked by direct sequencing (BGI Genomics, Beijing, China). To obtain recombinant human CXCL16 proteins, CHO cells were cultured in a six-well plate (F12 medium with 10% FCS, penicillin-streptomycin, 3 mM glutamine). After washing was performed, CHO cells were cultured in serum-free medium and transfected with 4 μg of CXCL16-I123A181, I123V181, T123A181, or T123V181 plasmids. Transfected cells were cultured in serum-free medium for 48 h. According to the manufacturer’s instructions, the supernatants were subsequently purified by a His-bind purification kit (Merck-Millipore, Burlington, MA, United States) to obtain purified CXCL16-I123A181, I123V181, T123A181, and T123V181 proteins.

GmSnRK1.1 was cloned into the pET29b (+) expression vector (Merck Millipore, United States), while GmWRKY31 was cloned into the pGEX-4T-1 expression vector (GE Healthcare, United States). The His-GmSnRK1.1 and glutathione S-transferase-GmWRKY31 proteins were separately produced in E. coli BL21 (DE3) cells, then harvested and purified using a GST-Sefinose kit (Sangon, China) or a His-bind Purification Kit (Merck Millipore). The pull-down assay was performed as described by Yang et al. (2008) (link), with minor modifications. In a total volume of 1 mL GST binding buffer (Sangon), the GST or GmWRKY31-GST recombinant proteins were incubated for 1 h at 4°C with 400 μL GST resin (Sangon), after which equal volumes of the GmSnRK1.1-His recombinant protein were added and incubated for 6 h at 4°C. The binding reaction was washed five times with binding buffer, each for 10 min at 4°C, then the pulled-down proteins were eluted by boiling, separated on a 12% SDS-PAGE gel, and immunoblotted with anti-His antibody and anti-GST antibody (Abmart, United States).

Heterologous expression, extraction, and purification of SFB-mFliC3, SFB-rFliC3, SFB-m5i-FliC3, and sal-FliC3 were performed as described elsewhere (31 (link)). Briefly, the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) was used to extract rat and mouse bacterial genomic DNA. The SFB-specific PCR primers 779F and 1008R were used to detect SFB DNA (32 (link)). Furthermore, on the basis of the conserved region in the SFB flagellin gene sequences obtained in our previous study, we designed a pair of SFB fliC3-specific primers, fliC3 F and fliC3 R. SFB fliC3 genes were subcloned into the pET-28a vector. Then, IPTG (Sangon Biotech, China) was added to overexpress FliC3 proteins in chemically competent BL21(DE3) cells (Transgen Biotech, Beijing, China). BugBuster master mix (Merck Millipore, Germany) was used to extract total bacterial proteins, a His Bind purification kit (Merck Millipore, Germany) was employed to purify SFB FliC3 proteins, and Pierce™ High Capacity Endotoxin Removal Spin Columns (Thermo Scientific™, USA) were used to eliminate endotoxins in the protein samples. A bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, China) was used to determine the protein concentrations.