title: Expression, purification of IL-38 in Escherichia coli and production of polyclonal antibodies.
abstract: 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 β-D-1-thio galactoside (IPTG) at 37°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.
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Zhonglan
authors.initials: Z
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Nongyu
authors.initials: N
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Xiu
authors.initials: X
authors.lastname: Teng
authors.affiliation: State Key Laboratories of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing 100193, People's Republic of China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Xiaoqiong
authors.initials: X
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Ke
authors.initials: K
authors.lastname: Qin
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Xueping
authors.initials: X
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Huan
authors.initials: H
authors.lastname: Tang
authors.affiliation: State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Cardiovascular Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, People's Republic of China. Electronic address: cuikaijun@sohu.com.
authors.forename: Kaijun
authors.initials: K
authors.lastname: Cui
authors.affiliation: 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.
authors.forename: Jiong
authors.initials: J
authors.lastname: Li
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007378:Interleukins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; C571243:IL-38 protein, human; D007378:Interleukins; D011993:Recombinant Fusion Proteins
keywords: Fusion protein; IL-38; Polyclonal antibodies