title: A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro.
abstract: As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Di
authors.initials: D
authors.lastname: Liu
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.affiliation: School of Pharmacy, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Wenyun
authors.initials: W
authors.lastname: Zheng
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Guo
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Wang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China.
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China. pwang11@ecust.edu.cn.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor engineering, East China University of Science and Technology, Shanghai, 200237, China. xymy@ecust.edu.cn.
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Ma
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D018928:Immunity, Mucosal; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014612:Vaccines / Q000235:genetics* / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007074:Immunoglobulin G; D050505:Mutant Proteins; D014612:Vaccines; C027538:heat-labile enterotoxin, E coli
keywords: Artificially assisted self-assembly; Heat-labile enterotoxin (LT); In vitro preparation; Mucosal adjuvant