Title
High Yield Expression of Recombinant CD151 in E. coli and a Structural Insight into Cholesterol Binding Domain
Author
Gayathri Purushothaman, Vijay Thiruvenkatam
Year
2019
Journal
Molecular Biotechnology
Abstract
CD151 is an abundantly expressed eukaryotic transmembrane protein on the cell surface. It is involved in cell adhesion, angiogenesis and signal transduction as well in disease conditions such as cancer and viral infections. However, the molecular mechanism of CD151 activation is poorly understood due to the lack of structural information. By considering the difficulties in expressing the membrane protein in E. coli, herein we introduce the strategic design for the effective expression of recombinant CD151 protein in E. coli with high yield, that would aid for the structural studies. CD151 having four transmembrane domain (TMD’s) along with small and a large extracellular loop (LEL) is constructed in parts to enhance the soluble expression of the protein attached with fusion tag. This has led to the high yield of the recombinant CD151 protein in the designed constructs. The recombinant CD151 protein is characterized and confirmed by western blot, CD and Mass peptide fingerprint. The molecular dynamics simulations (MDS) for the full-length CD151 shows conformational changes in the LEL of the protein in the presence and absence of cholesterol and indicate the certainty of closed and open conformation of CD151 based on cholesterol binding. The MDS results have led to the understanding of the possible underlying mechanism for the activation of the CD151 protein.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Protein folding, Biochemistry