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Abstract

Grant Number:	1R01GM081819-01
Project Title:	Integral membrane protein overexpression using organ bioreactors

PI Information:	Name	Email	Title
	ALLEN, PAUL D.	allen@zeus.bwh.harvard.edu	PROFESSOR

Abstract: DESCRIPTION (provided by applicant): The object of this research project is to produce functional recombinant mammalian integral membrane proteins (IMPs) in sufficient quantities to be useful for structural biology studies (X-ray, 2D electron crystallography and Single Particle Cryo-Electron microscopy). Our hypothesis is that to accomplish this goal it will be necessary to utilize the intrinsic protein folding capacity of mammalian cells to produce the purified IMPs needed for a detailed study of their native conformation. Membrane proteins comprise a very substantial proportion - up to 33% - of the predicted proteins of all organisms examined so far. They play a crucial role in many cellular and physiological processes such as acting as essential mediators of material and information transfer between cells and their environment, between compartments within cells, and between compartments comprising organ systems. In addition membrane proteins are vital to health and are already the target of the vast majority of drugs currently in use. An understanding of the structure of membrane proteins, however, represents only tiny fraction of the number of solved structures to date. Such structural information could significantly not only provide a basis for structure function analysis of membrane protein function but could facilitate the efficiency of drug discovery. Thus so far, most membrane protein structures have been solved for proteins that can be obtained from naturally rich sources. However, many of the proteins of greatest human physiological and pharmaceutical relevance are of relatively low abundance. The SPECIFIC AIM of this proposal is to devise a method to efficiently produce functional, properly folded mammalian IMPs in quantities sufficient to conduct high-resolution structural studies. To accomplish this aim we will test two novel parallel approaches: 1. Transgenic expression of a recombinant protein with a biotin acceptor domain (BAD) can both increase the efficiency of isolation of functional IMPs and multiple subunit IMP complexes sufficiently to use "native" organ expression or can be used to inducibly "over-express" IMPs in muscle and liver as in vivo organ bioreactors. 2. Direct acute expression of a recombinant IMP with a biotin acceptor domain (BAD) in muscle and liver as in vivo organ bioreactors of Wt animals can be used to directly or inducibly "over-express" IMPs for structural analysis. As proof of principle, we will express RyR1, a 2.2MDa homotetrameric ER protein, a 445Kda truncated C-terminal RyR1, and Cav1.1, the 5 subunit skeletal muscle slow voltage gated Ca2+channel.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
bioreactor, membrane protein, protein folding, protein purification, protein structure, recombinant protein, technology /technique development
NIH Roadmap Initiative tag, laboratory mouse, laboratory rat

Institution:	BRIGHAM AND WOMEN'S HOSPITAL
	RESEARCH ADMINISTRATION
	BOSTON, MA 02115
Fiscal Year:	2007
Department:
Project Start:	15-SEP-2007
Project End:	31-JUL-2010
ICD:	NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
IRG:	ZRG1

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