Ni nta resin column

For the purposes of this study, the mSEB (L45R, Y89A, Y94A) nucleotide sequences were synthesized by Beijing Genomics Institute (Shenzhen, China). Subsequently, mSEB was amplified via polymerase chain reaction (PCR). The primers utilized were as follows: forward primer (5‘-aaagagctcgagagtcaaccagatccta-3’) and a reverse primer: (5‘-agactcgagtcactttttctttgtcgtaagat3-’) with restriction sites for SacI and XhoI (underlined), allowing amplified DNA to be cloned into a pET28a expression plasmid (Merck, Germany) and transformed into Escherichia coli BL21. All recombinant plasmids were identified via DNA sequencing. The mSEB sequences have been submitted to GenBank (accession no. MT937070).
The mSEB proteins were produced via isopropylb-D-thiogalactopyranoside (IPTG) induction using pET28a-mSEB E. coli, as previously described (Zhou et al. 2008 (link)). The fusion protein was purified using a Ni-NTA resin column (Sangon Biotech, China) and Detoxi-Gel™ Endotoxin Removing Gel (Thermo, USA). Next, mSEB purified protein was verified using SDS-PAGE and western blotting.

The vector construction, expression, and purification of FGF protein were carried out as described previously. In brief, the coding sequence of FGF1 was amplified with specific primers (Table S1) and then inserted into the pET-32a vector (Novagen, Madison, WI, USA). After validation by sequencing, the recombinant vector pet32a-FGF1 was transformed into E. coli BL21 and cultured in 37 ℃ for 12 h. Then, the culture was diluted with LB (1:100) and incubated at 37 °C until OD value = 0.6 and then treated with isopropyl-β-D-thiogalactoside (0.01 mM) for 20 h at 16 °C. After centrifuging at 8000 rpm at 4 °C for 15 min, the bacterial cells were harvested, re-suspended in PBS, and disintegrated by ultrasonication on ice. After centrifuging at 12,000 rpm at 4 °C for 20 min, the supernatant was loaded onto a Ni-NTA resin column (Sangon, Shanghai, China). The column was then washed with PBS containing serial imidazole (20 mmol/L, 30 mmol/L, 50 mmol/L, 200 mmol/L, and 500 mmol/L). After verification using SDS-PAGE, the protein diluted in PBS containing 200 mmol/L and 500 mmol/L imidazole was collected. After removing imidazole by Ultrafiltration Spin Columns (Beyotime, Shanghai, China), the protein concentration was examined with BCA Protein Assay Kit (CWBIO, Taizhou, China) following standard protocols.