From Biomednet...
FASEB 2001 - Day 4 - Sunday 22 April 2001
Report:Get trendy, get folding Investigator: Rainer Rudolph
Sunday Apr 22nd, 2001
by Julie Clayton
If you want to know the structure and function of all those protein sequences, take a lesson from Rainer Rudolph, says a leading protein chemist today. Rudolph, Director of the Institute for Biotechnology at the Martin-Luther-University at Halle-Wittenberg in Germany, is one of the most successful entrepreneurs of protein science, who has combined intellectual creativity with industrial application. His approach might not be "trendy," said Chris Dobson, president of the Protein Society, "But now I think it's going to be the key science in developing how we can get all the structures and look at their functions in the future."
The developments Rudolph describes are "just the tip of the iceberg," according to Geoffrey Francis, of the Adelaide-based company GroPep, who develop candidate drug targets. "An important part of all pharmaceutical development is having the manufacturing processes to be able to produce recombinant material. For us, that involves having an efficient protein folding step," he told BioMedNet Conference Reporter.
Rudolph went literally from doing reactions in tiny Eppendorf tubes, to building his own giant industrial-scale fermentation tank, when he joined the German biochemicals giant Boehringer Mannheim to produce recombinant proteins in Escherichia coli.
The stumbling block, however, in all recombinant protein manufacture is the tendency for many proteins, particularly membrane-derived ones, to clump together as large protein aggregates called inclusion bodies, inside the bacterial cell cytoplasm.
These are "biologically inactive and useless," Rudolph told the audience.
This is because E. coli lacks the protein-folding machinery necessary to ensure correct folding of the nascent proteins, especially if they contain a number of disulphide bridges stabilizing the structure.
"This is a common problem that all of us share who use E. coli expression of recombinant proteins," said Francis. "Even in other systems, even in yeast, processors find themselves having to reprocess misfolded material, so you still have to have the skills of the protein folder to be able to make the overall manufacturing process efficient," he added.
Many growth factors and hormones have this propensity, and Rudolph has made a career out of solving the problem.
One such protein is tissue plasminogen activator (tPA), used as a "clot buster" drug, which has 17 disulphide bonds to recreate. Here, Rudolph achieved what many had said was impossible, by developing the correct conditions for folding. He isolated a new improved version of tPA from inclusion bodies, dissolved them, and then drove the correct folding in vitro, carefully controlling the temperature, and hence the rate of folding. He also discovered that by adding the amino acid arginine to the solution, the correct disulphide bonds would form. This massively improved the yield of the new tPA, which is now a successful clinical product.
Other proteins now benefiting from Rudolph's research are G-protein coupled receptors, the targets of many drugs now in development, and nerve growth factor (NGF). NGF is important in nervous-system development, and may have therapeutic potential in restoring the function of damaged nerves by neuroregeneration.
NGF has a particularly challenging tertiary structure to achieve in vitro, including one of three disulphide bonds that passes directly through a cysteine ring in the middle of the molecule. Rudolph and his team at the Martin-Luther-University at Halle-Wittenberg have added a different trick to their repertoire: inclusion of the 103 amino-acid pro-sequence of the growth factor, which is normally cleaved during synthesis to produce a 118 amino-acid mature protein. The pro-sequence itself acts as a folding partner. According to the most recent mass spectrometry results, the pro-NGF protein appears correctly folded, with the three disulphide-bridges intact, within ten minutes of the folding process.
The team are also improving the yield of proteins such as pro-insulin, to which the addition of a signal sequence ensures secretion into the E. coli endoplasm.
These successes could herald a renaissance in the field, according to Dobson, group leader at the New Chemistry Laboratory at the University of Oxford, who advises on many structural and functional genomics projects.
"It's absolutely clear that the number one bottle neck at the moment is producing protein and refolding it. Once you've got the protein refolded, particularly if it's a membrane protein, once you have those pure, then the rest of it is relatively easy, because we can apply all these structural methods," he said... |
