August 2017  Vol. 27  No. 8 J. Microbiol. Biotechnol. (2017), 27(8), 1461–1471 https://doi.org/10.4014/jmb.1704.04006 Research Article jmb Review Elucidating Bottlenecks to the Efficient Preparation of AB 5 -Hexamer Mucosal Adjuvant Protein LTm by Genetic Engineering Di Liu 1 , Fabiao Hu 1 , Wenpeng Wang 1 , Dong Wu 1 , Xiujuan He 2 , Wenyun Zheng 2 * , Haipeng Liu 3 * , and Xingyuan Ma 1 * School of Biotechnology and State Key Laboratory of Bioreactor Engineering, School of Pharmacy, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai 200237, P.R. China State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, P.R. China Introduction Heat-labile enterotoxin (LT) is an AB -type bacterial toxin produced by enterotoxigenic Escherichia coli [1]. The complete hexameric structure comprises one A subunit (LTA) (27 kDa) and a ring-like pentamer formed by five B subunits (LTB) (11.6 kDa each) [2, 3]. Both the LTA and LTB subunits contain 18 and 21 amino-acid-long signal peptides at the N-terminus after translation [4, 5]. Sequence analysis revealed that LT subunit-encoding genes, eltA and eltB, are in one operon, and both genes are transcribed as a single mRNA molecule. The open reading frames of eltA and eltB overlap, with a translational coupling between them [6]. After synthesis in the cytoplasm, both the LTA and LTB subunits translocate across the bacterial cytoplasmic membrane into the periplasmic space under the guidance of signal peptides. There, the leader of the subunits is removed and the AB complex is assembled [7]. As a bacterial toxin, the LT non-toxic B subunit pentamer has the ability to bind to GM gangliosides, which are expressed on the surface of many cell types; the LT toxic A subunit has ADP-ribosyltransferase activity, which causes Received: April 6, 2017 Revised: May 10, 2017 Accepted: May 18, 2017 First published online May 24, 2017 *Corresponding authors W.Z. Phone: +862164253980; Fax: +862164253980; E-mail: zwy@ecust.edu.cn H.L. Phone: +865912183203; Fax: +865912183203; E-mail: haipengliu@xmu.edu.cn X.M. Phone: +862164250135; Fax: +862164250135; E-mail: xymy@ecust.edu.cn upplementary data for this paper are available on-line only at http://jmb.or.kr. pISSN 1017-7825, eISSN 1738-8872 Copyright © 2017 by The Korean Society for Microbiology and Biotechnology Escherichia coli heat-labile enterotoxin (LT) and its non-toxic mutant (LTm) are well-known powerful mucosal adjuvants and immunogens. However, the yields of these adjuvants from genetically engineered strains remain at extremely low levels, thereby hindering their extensive application in fundamental and clinical research. Therefore, efficient production of these adjuvant proteins from genetically engineered microbes is a huge challenge in the field of molecular biology. In order to explore the expression bottlenecks of LTm in E. coli, we constructed a series of recombinant plasmids based on various considerations and gene expression strategies. After comparing the protein expression among strains containing different recombinant plasmids, the signal sequence was found to be critical for the expression of LTm and its subunits. When the signal sequence was present, the strong hydrophobicity and instability of this amino acid sequence greatly restricted the generation of subunits. However, when the signal sequence was removed, abundantly expressed subunits formed inactive inclusion bodies that could not be assembled into the hexameric native form, although the inclusion body subunits could be refolded and the biological activity recovered in vitro. Therefore, the dilemma choice of signal sequence formed bottlenecks in the expression of LTm. These results reveal the expression bottlenecks of LTm, provide guidance for the preparation of LTm and its subunits, and certainly help to promote efficient preparation of this mucosal adjuvant protein. Keywords: Mucosal adjuvant LTm, AB-hexamer protein, expression bottlenecks, preparation strategies, genetic engineering S S