Current Methods for Membrane Protein Expression and Purification for Structure Solution Studies

TOPICAL COLLECTIONS

Guest Editor

Shamayeeta Ray

Harvard University, Department of Molecular and Cellular Biology

Dr. Shamayeeta Ray is a postdoctoral researcher in the Department of Molecular and Cellular Biology in Harvard University. She is an experienced structural biologist with extensive research experience and expertise in x-ray crystallography of membrane and soluble proteins, biochemistry, molecular and cell biology, and recombinant gene manipulation. Her current research focusses on understanding the molecular mechanism of transport of essential divalent transition metals by Nramp family of transmembrane transporters. For this work, she is using lipidic cubic phase-based X-ray methods along with biochemical and biophysical approaches to map various conformations and associated thermodynamics of the metal transport pathway of Nramps so that we can uncover the generic mode of transport and homeostasis which can help developing therapeutics for diseases related to metal overload or deficiency. She obtained her graduate degree from the Department of Chemistry of Indian Institute of Technology (IIT) Bombay, India where her graduate research focused on x-ray-based structure elucidation of regulatory proteins useful in developing biosensors for water purification system. Another of her graduate project involved structural characterization of a bacterial TetR family regulator, TylP, which controls the levels of the commercially important antibiotic tylosin within cells. With ten years of experience in structural biochemistry, she has mastered technical expertise in cloning, protein expression in bacterial and mammalian systems, protein production and crystallization, x-ray data collection and structure resolution. In addition to research, she has a passion for mentoring and teaching and is actively involved with organizations promoting STEM research for women.

Collection Overview

Membrane proteins are essential biological molecules as they play critical roles in various biological processes in all organisms. They aid in transporting various substrates such as ions, neurotransmitters, and essential amino acids into and out of cells, as well as participating in signal transduction and multidrug resistance. Due to their clinical significance, membrane proteins serve as targets for a host of different drugs.

Hence, to understand their functions and to develop drugs, it is essential to elucidate the three-dimensional structures of these proteins. Compared to soluble proteins, it is more challenging to obtain structures of membrane proteins as they need to be extracted from their native lipid environment and retain their correct conformation and folding in detergents and other membrane mimics. Moreover, membrane proteins are expressed with much lower yields, hindering structural studies.

Recent advances in structure elucidation techniques like cryo-EM and cryo-ET along with X-ray crystallography provide a broader spectrum for the structure determination process of membrane proteins.

In this methods section, we will explore the methods currently in practice for expressing and obtaining optimum yields of diverse membrane proteins for structural studies. Various expression systems will be used, from bacterial to insect to mammalian cells; membrane proteins, either alone or in complex with antibodies, and nanobodies that aid in increasing the overall mass for cryo-EM analysis are desired for structural research. Knowledge of various methodologies in a visual format, such as that provided by JoVE, can help streamline the process of obtaining a high yield of membrane proteins in stable forms in order to resolve high-resolution structures and will have an impact on the structural biology community in general.

Guest Editor

Collection Overview

Articles

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies