Proteins while you wait

Associate Professor Stephen Bottomley of Monash University's Department of Biochemistry and Molecular Biology will speak in the new technologies session at the Lorne conference on Protein Structure and Function this week.

In his presentation on the development of high-throughput protein expression technology to support biomedical research, he will describe a production pipeline for high-throughput cloning and expression of proteins now established at Monash University in Melbourne.

This facility is available to anyone who wants large-scale and rapid protein production, and is designed to speed up the entire process of protein production from gene to purified product including cloning, expression and purification.

It is also designed to use in combination with the protein analysis and crystallography facilities at Monash.

The facility has already produced more than 2000 individual proteins over the 18-month establishment phase of the pipeline, including around 400 proteins for vaccine trials. The initiative was initially funded by a new research initiatives grant from Monash University, an ARC Linkage grant and through the ARC Centre for Excellence in Structural and Functional Microbial Genomics at Monash.

It is a large collaborative effort led by Monash's Departments of Biochemistry and Microbiology together with scientists from the Walter and Eliza Hall Institute and Melbourne University.

Misfolding disorders

Bottomley has held a long-time research interest in the molecular basis of protein folding and misfolding, with the ultimate aim of preventing diseases associated with defects in protein folding, such as emphysema, Alzheimer's, cirrhosis and Huntington's disease.

His group studies two protein families involved in misfolding disorders: the serine proteinase inhibitor (serpin) and polyglutamine family of proteins. The ability of proteins to rapidly and correctly fold from a structureless state to their native conformation is remarkable and absolutely required for proper protein secretion and function.

That process, however, is poorly understood. Protein misfolding can lead to a lack of secretion or protein aggregation, both of which can cause damage to the normal functioning of tissues and therefore disease. Bottomley's group has used a multidisciplinary approach over many years to better understand the mechanisms underlying this process.

In fact, they were the first to kinetically and structurally characterise particular protein misfolding events and identify key steps in the process. Specifically, they determined the structure of a serpin polymer and more recently have identified novel conformational characteristics of the polyglutamine-family member, ataxin-3.

These studies will provide clues as to how some folding defects and subsequent damage can be overcome clinically.

High-throughput methods

Arising from this work and developed in parallel was the need for high-throughput methods for rapid, larger scale purification and production of recombinant proteins for both research and for the screening of candidate therapeutic molecules. This has become a major challenge for biotechnology in general and indeed for any laboratory requiring proteins for their research.

A major advance in high-throughput methods for proteins developed by Bottomley and his team (published in BMC Biotechnology in 2006) was development of a set of E. coli expression vectors to streamline the cloning, expression and purification steps of protein production.

These vectors overcame one of the main hurdles in establishing rapid protein purification, particularly on a large scale - that is, a way to express most mammalian proteins easily and inexpensively in an E. coli expression system, preferred by researchers but previously unsuccessful for the expression of many mammalian targets without considerably fiddling around with conditions. These vectors allowed for quick and easy parallel cloning and expression, suitable at the laboratory level and adaptable for high-throughput situations.

In addition, Bottomley and Monash colleague Ashley Buckle have developed REFOLD, a web database of published methods to assist in the design and implementation of methodologies for the refolding of recombinant proteins. This database has been used by over 50,000 scientists to help them refold their proteins.

In his presentation at Lorne, Bottomley will discuss how the abundance of genomic sequence data enables the application of automated methods for high-throughput production of proteins that are used for structural and functional studies.

The pipeline established at Monash "aims to establish a unified technological infrastructure suitable for use by large-scale structural biology initiatives but also by individual laboratories", Bottomley says. In his presentation, he will overview the pipeline's technological developments and present work from his own group on new tools for more reliable and efficient protein production.