Escherichia coli bl21

GenScript constructed the recombinant plasmid pGEX-4-T-1-rSPA containing the rSPA gene sequence (Nanjing, People’s Republic of China [PRC]). The plasmid (pET-44a, pET-30a, pCANTAB5E), Escherichia coli BL21, Ex Taq deoxyribonucleic acid (DNA) polymerase, deoxynucleotide triphosphates (dNTPs), and DNA-purification kit were purchased from Tiangen Biotech (Beijing, PRC). A DNA ligation Kit, DNA markers, and restriction endonucleases were obtained from Takara Biotech (Dalian, PRC). Our laboratory prepared the rabbit antialpaca IgG antibody. Isopropyl-β-d-thiogalactoside (IPTG), horseradish peroxidase (HRP)-conjugated goat antirat IgG, and anti-His monoclonal antibody were provided by Beijing Biosynthesis Biotechnology (Beijing, PRC). Quartz crystal microbalance (QCM) measurements were performed using a Q-Sense D300 instrument with 5 MHz quartz crystals deposited with gold electrodes (14 mm diameter) on a single side of the surface (Q-Sense, Gothenburg, Sweden). All other reagents were at least analytical grade for this study, and were purchased from Sigma-Aldrich (St Louis, MO, USA).

To obtain the GST fusion proteins of Smurf1, the DNA fragment of Smurf1 was subcloned into pGEX-4T-1 vector (GE Healthcare). GST and GST-fusion proteins were expressed in Escherichia coli BL21 (Tiangen Biotech) and purified with glutathione-Sepharose 4B beads (Pharmacia Biotech). His-MBP-Kindlin-2 was expressed in E. coli BL21 and purified with His-Select HF Nickel Affinity Gel (Sigma-Aldrich). To detect the direct binding of Kindlin-2 with Smurf1, GST or GST-smurf1 was immobilized on GST 4B beads and washed, then beads were incubated with His-MBP-Kindlin-2 purified by MBP Affinity Matrix (Amylose Resin; New England Biolabs, Inc.) or His-Select HF Nickel Affinity Gel for 12 h at 4°C under rotation. Bacterial-expressed His-MBP-Kindlin-2 bound to MBP Affinity Matrix or His-Select HF Nickel Affinity Gel was incubated with GST-Smurf1 or GST for 12 h at 4°C. Then beads were washed with RIPA buffer three times, and proteins were eluted, followed by Western blot analysis.