Expression and Purification of Recombinant Truncated IL-8

The expression and purification of recombinant truncate IL-8 was conducted as described in our previous study57 (link). Briefly, the plasmid T-tIL-8 was digested with EcoRI and XhoI and the resultant products were inserted into the EcoRI/XhoI-digested pET32a (+) vector to construct the recombinant expression plasmid, named as P-tIL-8. The plasmid was then transformed into E. coli BL21 and induced by adding 1.0 mM IPTG at 37 °C for 4 h. the cells were centrifuged at 8000× g for 10 min at 4 °C and suspended with sterile phosphate buffer saline (PBS), followed by ultrasonicating with an ultrasonic cell disrupter (JY92-IIDN, Ningbo Scientz, China) and examined by 12.5% SDS-PAGE. The inclusion bodies from the insoluble fractions were purified by Ni-NTA-Sefinose Column (Sangon Biotech, Shanghai, China) after dissolution in 8 M urea solutions and filtration with 0.22-μm filters. The refolding of the purified proteins was conducted by dialyzing gradiently from 6 M urea solutions to PBS at 4 °C, and then analyzed by 12.5% SDS-PAGE. The protein was quantified using the Bradford assay with bovine serum albumin (BSA) as standard and a NanoDrop spectrophotometer (Thermo Scientific) according to the manufacturer’s instructions. Purified proteins were named as rtIL-8 and stored at 20 °C.