PMID- 25448591 OWN - NLM STAT- MEDLINE DCOM- 20150831 LR - 20141217 IS - 1096-0279 (Electronic) IS - 1046-5928 (Linking) VI - 107 DP - 2015 Mar TI - Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies. PG - 76-82 LID - S1046-5928(14)00250-2 [pii] LID - 10.1016/j.pep.2014.10.016 [doi] AB - Members of the interleukin-1 (IL-1) family play important roles in inflammation and host defense against pathogens. Here, we describe a novel member of the IL-1 family, interleukin-38 (IL-38, IL-1F10, or IL-1HY2), which was discovered in 2001. Although the functional role of IL-38 remains unclear, recent reports show that IL-38 binds to the IL-36 receptor (IL-36R) which is also targeted by the IL-36 receptor antagonist (IL-36Ra). Consequently, these two molecules have similar effects on immune cells. Here, we describe the expression of soluble and active recombinant IL-38 in Escherichia coli (E. coli). The IL-38 gene sequence was optimized for expression in E. coli and then cloned into a pEHISTEV expression vector, which has an N-terminal 6-His affinity tag under control of the T7 lac strong promoter. Optimization of culture conditions allowed induction of the recombinant fusion protein with 0.1 mM isopropyl beta-D-1-thio galactoside (IPTG) at 37 degrees C for 4h. The recombinant fusion protein was purified using an Ni affinity column and was further digested with TEV protease; the cleaved protein was purified by molecular-exclusion chromatography. Next, we measured IL-38 binding ability using functional ELISA. The purified proteins were used to immunize a New Zealand white rabbit four times to enable the production of polyclonal antibodies. The specificity of the prepared polyclonal antibodies was determined using Western blot, and the results showed they have high specificity against IL-38. Here, we describe the development of an effective and reliable method to express and purify IL-38 and anti-IL-38 antibodies. This will enable the function and structure of IL-38 to be determined. CI - Copyright (c) 2014 Elsevier Inc. All rights reserved. FAU - Hu, Zhonglan AU - Hu Z AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Chen, Zhenyu AU - Chen Z AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Huang, Nongyu AU - Huang N AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Teng, Xiu AU - Teng X AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Zhang, Jun AU - Zhang J AD - State Key Laboratories of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, People's Republic of China. FAU - Wang, Zhen AU - Wang Z AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Wei, Xiaoqiong AU - Wei X AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Qin, Ke AU - Qin K AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Liu, Xiao AU - Liu X AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Wu, Xueping AU - Wu X AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Tang, Huan AU - Tang H AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Zhu, Xiaofeng AU - Zhu X AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. FAU - Cui, Kaijun AU - Cui K AD - Department of Cardiovascular Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. Electronic address: cuikaijun@sohu.com. FAU - Li, Jiong AU - Li J AD - State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. Electronic address: lijionghh@sina.com. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20141106 PL - United States TA - Protein Expr Purif JT - Protein expression and purification JID - 9101496 RN - 0 (Antibodies) RN - 0 (IL-38 protein, human) RN - 0 (Interleukins) RN - 0 (Recombinant Fusion Proteins) SB - IM MH - Animals MH - Antibodies/*analysis/immunology MH - Blotting, Western MH - Cloning, Molecular MH - Escherichia coli/*genetics/metabolism MH - *Gene Expression MH - Interleukins/genetics/immunology/*isolation & purification MH - Rabbits MH - Recombinant Fusion Proteins/genetics/immunology/isolation & purification OTO - NOTNLM OT - Fusion protein OT - IL-38 OT - Polyclonal antibodies EDAT- 2014/12/03 06:00 MHDA- 2015/09/01 06:00 CRDT- 2014/12/03 06:00 PHST- 2014/08/08 00:00 [received] PHST- 2014/10/28 00:00 [revised] PHST- 2014/10/30 00:00 [accepted] PHST- 2014/12/03 06:00 [entrez] PHST- 2014/12/03 06:00 [pubmed] PHST- 2015/09/01 06:00 [medline] AID - S1046-5928(14)00250-2 [pii] AID - 10.1016/j.pep.2014.10.016 [doi] PST - ppublish SO - Protein Expr Purif. 2015 Mar;107:76-82. doi: 10.1016/j.pep.2014.10.016. Epub 2014 Nov 6.