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 Ca2+-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.

High yields and soluble expression of superoxide dismutases in Escherichia coli due to the HIV-1 Tat peptide via increases in mRNA transcription

“…Preliminary experimental data showed that Tat-LSECTin was mainly expressed in the cell lysate supernatant, in the form of inclusion bodies, and that the Tat-LSECTin protein expression yield was significantly higher than that of LSECTin alone. 41 These results support the idea that Tat core peptides also promote high yield and soluble expression of membrane proteins containing disulfide bonds, and indicate that addition of a Tat tag could promote high yield and soluble expression of proteins in E. coli regardless of whether they were membrane proteins or disulfide bond-rich proteins, thereby providing a novel tool for heterologous protein expression in E. coli .…”

Novel Chitin Deacetylase from Thalassiosira weissflogii Highlights the Potential for Chitin Derivative Production

“…These transmembrane helix structures may cause difficulties for our protein expression in vitro. However, producing a prokaryotic membrane protein in the simple, flexible, and inexpensive E. coli system has already proven successful in many cases [ 55 , 56 , 57 ]. Unfortunately, the yield and quality of membrane proteins, especially eukaryotic ones, are often insufficient for structural and functional studies in E. coli [ 58 ].…”