Imidazole

The N-terminal of mouse CYLC1 was cloned into the pET-N-His-C-His vector (Beyotime, Shanghai, China) and then transfected into the ER2566 E. coli strain (Weidi Biotechnology, Shanghai, China). CYLC1 protein expression was induced by 1 mM IPTG (Beyotime) at 24°C for 6 hr. After centrifugation, the bacterial pellet was resuspended in buffer 1 (50 mM Tris–HCl pH 8.0, 200 mM NaCl), and the proteins were released by sonication. After centrifugation, anti-His beads (Beyotime) were added to the supernatant and incubated for 2 hr. After washing five times with buffer 1, CYLC1-N protein was eluted with 250 mM imidazole (Beyotime). The protein was concentrated by centrifugation with ultrafiltration centrifuge tube (Millipore, Shanghai, China). One hundred micrograms of CYLC1-N protein was emulsified at a 1:1 ratio (vol/vol) with Freund’s complete adjuvant (Beyotime) and administered subcutaneously into New Zealand white rabbits (Charles River) at multiple points. For the subsequent three immunizations, 50 μg CYLC1-N protein was emulsified with incomplete Freund’s adjuvant (Beyotime) at an interval of 2 weeks. One week after the last immunization, blood was collected, and the serum was separated.

Full-length mouse TEKTIP1 was cloned into the pET-N-His-C-His vector (Beyotime, Shanghai, China) and then transfected into the ER2566 E. coli strain (Weidi Biotechnology, Shanghai, China). Protein expression was induced by 1 mM IPTG (Beyotime) at 30 ℃ overnight. After centrifugation, the bacterial pellet was resuspended in buffer (50 mM Tris–HCl pH 8.0, 200 mM NaCl), and the proteins were released by sonication. After centrifugation, anti-His beads (Beyotime) were added to the supernatant and incubated overnight at 4 ℃. After washing, recombinant protein was eluted with 250 mM imidazole (Beyotime). Coomassie brilliant blue stain of the gel of purified TEKTIP1 was shown in Supplementary Fig. 6. Recombinant TEKTIP1 protein was emulsified at a 1:1 ratio (v/v) with Freund’s complete adjuvant (Beyotime) and administered subcutaneously into ICR female mice at multiple points. For the subsequent three immunizations, recombinant TEKTIP1 protein was emulsified with incomplete Freund’s adjuvant (Beyotime) at an interval of 2 weeks. One week after the last immunization, blood was collected, and the serum was separated.