title: Tat peptide-mediated soluble expression of the membrane protein LSECtin-CRD in Escherichia coli.
abstract: The human liver and lymph node sinusoidal endothelial cell C-type lectin (hLSECtin), a type II integral membrane protein, containing a Ca(2+)-dependent carbohydrate recognition domain (CRD), has a well-established biological activity, yet its three-dimensional structure is unknown due to low expression yields and aggregation into inclusion bodies. Previous study has demonstrated that the HIV-1 virus-encoded Tat peptide ('YGRKKRRQRRR') can increase the yields and the solubility of heterologous proteins. However, whether the Tat peptide could promote the high-yield and soluble expression of membrane proteins in Escherichia coli is not known. Therefore, the prokaryotic expression vector pET28b-Tat-hLSECtin-CRD (using pET28b and pET28b-hLSECtin-CRD as controls) was constructed, and transformed into E. coli BL21 (DE3) cells and induced with isopropyl-β-d-thiogalactoside (IPTG) followed with identifying by SDS-PAGE and Western blot. Subsequently, the bacterial subcellular structure, in which overexpressed the heterologous proteins Tat-hLSECtin-CRD and Tat-free hLSECtin-CRD, was analyzed by transmission electron microscope (TEM) respectively, and the mannose-binding activity of Tat-hLSECtin-CRD was also determined. Expectedly, the solubility of Tat-LSECtin-CRD significantly increased compared to Tat-free LSECtin-CRD (**p < 0.01) with prolonged time, and the Tat-LSECtin-CRD had a significant mannose-binding activity. The subcellular structure analysis indicated that the bacterial cells overexpressed Tat-hLSECtin-CRD exhibited denser region compared with controls, while dot denser region aggregated in the two ends of bacterial cells overexpressed Tat-free hLSECtin-CRD. This study provided a novel method for improving the soluble expression of membrane proteins in prokaryotic systems by fusion with the Tat peptide, which may be potentially expanded to the expression of other membrane proteins.
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Guofu
authors.initials: G
authors.lastname: Dong
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Changzhen
authors.initials: C
authors.lastname: Wang
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, P. R. China.
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Jianbo
authors.initials: J
authors.lastname: Cong
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Cheng
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Mingqun
authors.initials: M
authors.lastname: Wang
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Pengkai
authors.initials: P
authors.lastname: Zhao
authors.affiliation: Beijing Institute of Radiation Medicine, Department of Genomics and Proteomics, Chinese Human Genome Center, Beijing, P. R. China.
authors.forename: Li
authors.initials: L
authors.lastname: Tang
authors.affiliation: Beijing Institute of Radiation Medicine, State Key Laboratory of Proteomics, Cognitive and Mental Health Research Center of PLA, Beijing, P. R. China.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Beijing Institute of Radiation Medicine, Beijing, P. R. China.
authors.forename: Ke
authors.initials: K
authors.lastname: Wu
mesh_terms: D050260:Carbohydrate Metabolism / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008358:Mannose / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D037181:Lectins, C-Type; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; C497137:tat peptide (47-57), Human immunodeficiency virus 1; D008358:Mannose