To express and purify the recombinant LN- and FN-binding proteins, S. suis strain ZY05719 genomic DNA was used as template for PCR using the primers listed in Table 1 , with PrimeSTAR HS DNA polymerase (Vazyme). A total of nine PCR products were inserted into the pET32a or pET28a vectors using the XhoI and BamH I sites and transformed into E. coli BL21 (DE3) for expression at 37°C. The cloned sequences were confirmed by DNA sequence analysis. Recombinant proteins were induced with 1 mM IPTG at 37°C for 5 h when the cell had grown to an OD600 of 0.6. Bacterial cultures were harvested by centrifugation at 4°C for 10 min at 12,000 × g. The induced proteins were purified from cultures by Ni-chelating chromatography (GE Healthcare) according to the manufacturer's recommendations.
Ni chelating chromatography
Manufactured by GE Healthcare
Ni-chelating chromatography is a purification technique used to separate and purify proteins and other biomolecules. It utilizes a solid support matrix, such as agarose beads, with immobilized nickel ions (Ni2+) that can bind to proteins containing specific amino acid sequences, typically histidine tags. This method allows for the selective capture and isolation of the target protein from a complex mixture, enabling its subsequent analysis or further processing.
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Lab products found in correlation
4 protocols using ni chelating chromatography
1
Recombinant Protein Expression and Purification
2
Cloning and Purification of Recombinant EF-Tu
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The ef-tu gene encoding one of the most important novle virulence associated factors was cloned into pET-32a using homologous recombination technology (ClonExpress®IIOne Step Cloning Kit, Vazyme Biotech Co., Ltd). The sequence that overlapped with the end of the cloning site was added onto the insert through a PCR step. Table 1 lists the primers used in this study. The reconstructed plasmid was transformed into Escherichia coli BL21 (DE3) for isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression, and the induced proteins were purified by Ni-chelating chromatography (GE Healthcare). Polyclonal antibody against the recombinant EF-Tu (rEF-Tu) was prepared by subcutaneously immunizing 1-month-old New Zealand white rabbits. Each rabbit was immunized a total of three times with 1 mg of purified recombinant protein emulsified in Freund's adjuvant (Sigma, USA) at 2-week intervals. Sera were collected 1 week after the third immunization. The animals used in this study met the legal and ethical requirements and were treated humanely.
3
Mapping the Binding Regions of MRP Protein
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To determine the binding region of MRP, four recombinant fragments of MRP (Fig. 1A ) were engineered mainly according to its predicted secondary structure (Supplementary Fig. 1B ) as follows: MRP-N (a.a. 48–721, the N-terminus of MRP), including MRP-N1 (a.a. 48–282, the α-coil-rich region), MRP-N2 (a.a. 283–721, mainly consisting of β-sheets) and MRP-C (722–1257, a proline-rich region mainly consisting of β-sheets). The oligonucleotide primers and expression vector used for MRP-N, MRP-N1, MRP-N2 and MRP-C in this study are listed in Supplementary Table 1 . All of the above ORFs were cloned into the pET-28a vector, and the proteins were expressed and purified by Ni-chelating chromatography (GE Healthcare) according to the manufacturer’s recommendations.
4
Recombinant OmpA and CME Protein Expression and Antibody Production
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To express His-tag recombinant outer membrane protein (OmpA) and seven CMEs, namely, acetate kinase (AckA), fructose-bisphosphate aldolase (FbaA), fumarate reductase flavoprotein subunit (FrdA), L-lactate dehydrogenase (LDH), dihydrolipoamide dehydrogenase (LpdA), pyruvate dehydrogenase (Pdh), phosphoglycerate kinase (Pgk), and phosphoenolpyruvate synthase (PpsA), the ExPEC strain RS218 genomic DNA was used as the template for PCR using the primers listed in Table 2
Antibodies preparation was completed by Shanghai Willget Biotechnology Co., Ltd. The peptides used for immunization were designed and synthesized based on the amino acid sequence of CMEs and OmpA. Rabbits were immunized with peptides to obtain serum, and then specific antibodies were obtained by affinity purification of the antigen.Supplementary Table S1
Antibodies preparation was completed by Shanghai Willget Biotechnology Co., Ltd. The peptides used for immunization were designed and synthesized based on the amino acid sequence of CMEs and OmpA. Rabbits were immunized with peptides to obtain serum, and then specific antibodies were obtained by affinity purification of the antigen.
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