title: Soluble expression and purification of the functional interleukin-30 protein in Escherichia coli.
abstract: Interleukin-30 (IL-30), or IL-27p28, is the α subunit of IL-27 constructed by Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28 binding via noncovalent bonds. IL-30 can be independently secreted and function independently of IL-27. Recent studies demonstrated IL-30 could concurrently antagonize T helper 1 (Th1) and Th17 responses and might have therapeutic implications for controlling autoimmune diseases. However, no reports have stated an efficient method to generate a relatively large quantity of IL-30. In this study, an Escherichia coli expression system for the rapid expression of the mouse IL-30 is developed. For the first time, IL-30 was expressed in a form of soluble fusion protein and purified using a method of simple affinity chromatography. In order to avoid the impact of minor codons on expressing eukaryotic protein in E. coli and to improve the expression quantity, the nucleotide sequence of IL-30 was optimized. The optimized gene sequence was then subcloned into the pET-44a(+) vector, which allowed expression of IL-30 with a fusion tag, NusA. The vector was transformed into E. coli and the expressed fusion protein, NusA-IL-30, was purified by Ni chromatography. Then the fusion tag was removed by cleavage with thrombin. The purity of purified IL-30 was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as high-performance liquid chromatography (HPLC) and the purity was up to about 92%. The yield of IL-30 was 8.95 mg from 1 L of bacterial culture. Western blot confirmed the identity of the purified protein. The recombinant IL-30 showed its biological activity by inhibiting Th17 differentiating from naive CD4(+) T cells. Therefore, this method of express and purifying IL-30 provides novel procedures to facilitate structural and functions studies of IL-30.
authors.affiliation: a State Key Laboratories of Agrobiotechnology, College of Biological Sciences , China Agricultural University , Beijing , China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Liu
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Nongyu
authors.initials: N
authors.lastname: Huang
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Zhonglan
authors.initials: Z
authors.lastname: Hu
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Wenling
authors.initials: W
authors.lastname: Wu
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Xiu
authors.initials: X
authors.lastname: Teng
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Xiaoqiong
authors.initials: X
authors.lastname: Wei
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Huan
authors.initials: H
authors.lastname: Tang
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Xueping
authors.initials: X
authors.lastname: Wu
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Chen
authors.affiliation: b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy , West China Hospital, Sichuan University , Chengdu , Sichuan , China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Li
authors.affiliation: a State Key Laboratories of Agrobiotechnology, College of Biological Sciences , China Agricultural University , Beijing , China.
authors.forename: Zandong
authors.initials: Z
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007113:Immunity, Innate; D007378:Interleukins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D058504:Th17 Cells / Q000166:cytology / Q000187:drug effects
chemical_list: D007378:Interleukins; D011993:Recombinant Fusion Proteins
keywords: Autoimmune diseases; IL-30; Th1/Th17; high yield and high purity; soluble expression and purification