GE-HEALTHCARE Purifying Challenging Proteins: Membrane proteins, Multiprotein complexes and Inclusion bodies

protein purification

Introduction

This handbook is intended for students and experienced researchers with an interest in the isolation of integral membrane proteins, multiprotein complexes, or in refolding proteins from inclusion bodies. The aim is to present tools, strategies, and solutions available to meet the purification challenges associated with these three classes of proteins.For a background on techniques for protein purification, in general and handling recombinant proteins, we recommend the Recombinant Protein Purification Handbook and other handbooks in this series (see “Related literature” on page 100).

Challenging proteins

Our knowledge and understanding of the structural and functional biology of soluble proteins has increased dramatically over the last decade. Much of the technology for the production, purification, and analysis of soluble proteins is now at a stage where generic purification protocols allow relatively high success rates.The situation is different for the areas that this handbook covers; integral membrane proteins, multi protein complexes, and inclusion bodies. The need to handle and study these more difficult groups of proteins is clear, given that

• About 30% of a typical cell’s proteins are membrane proteins, and more than 50% of the current drugs on the market exert their actions via membrane proteins

• While carrying out their enzymatic, structural, transporting, or regulatory functions, proteins most often interact with each other, forming multiprotein complexes

• A large proportion of normally soluble proteins that are overexpressed in E. coli end up as incorrectly folded and insoluble protein in inclusion bodies

Outline

After a general introduction to each area, high-level consensus workflows are presented to summarize current best practices in each area. Rather than providing a number of detailed protocols that have been optimized for individual proteins, this handbook provides general advice or generic protocols in a step-by-step format. The generic protocols are intended as starting points for development of separation protocols. Details will typically have to be changed to tailor the protocols for individual proteins. Furthermore, the required variations to the generic protocols cannot be predicted and unless appropriate changes are made, the protocols will only work poorly, if at all—this is one of the major challenges for the researcher involved with these groups of proteins. To address this issue, the generic protocols are presented with critical parameters identified, together with ranges of values to test for the parameters. The handbook also provides guidance, hints, and tips when using protocols other than those described here.

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