Designing Proteins for Crystallization and Optimizing Protein Expression using Whole Gene Synthesis

Dr. Alex Burgin, Director of Molecular Biology, deCODE biostructures, speaking at Protein Crystallography in Drug discovery 2005.

Abstract

It is clear that some modifications to a protein (deletions, fusions, point mutations, etc.) can have very little effect on the structure of a protein but can have profound effects on expression, solubility and crystallization. Unfortunately, the best modification(s) may be difficult to identify de novo and therefore usually requires generating and testing multiple protein constructs.

In addition, these constructs can be difficult to make using traditional site directed mutagenesis methods since it may require removing internal domains/loops or generating a large number of different mutations across the gene. To overcome this fundamental problem we have developed whole gene synthesis technology that allows the rapid generation of multiple gene variants for expression and crystallization trials. This approach is facilitated by a database and algorithm software package that aids in the design of protein and gene constructs for X-ray crystallography.

The software allows the user to define protein constructs based on crystal structures of homologous proteins, phylogenetic analyses, and protein prediction algorithms. The software also designs oligonucleotides that are required to synthesize the genes by PCR. Factors which affect expression (codon usage, mRNA secondary structure, etc.) are incorporated into the gene design which also improves protein expression. Examples of how we have used the application to design multiple protein constructs that crystallize more readily and diffract X-rays to higher resolution will be presented.

Launch Presentation

About the speaker


Dr. Burgin received his Ph.D. in genetics at Indiana University in 1990, and then completed post-doctoral work at the National Institutes of Health focusing on bio-organic chemistry and enzyme mechanism. He joined Ribozyme Pharmaceuticals in 1994 as a Senior Scientist, and became Group Leader in 1995. At Ribozyme, Dr. Burgin oversaw the development of synthetic ribozymes with improved catalytic activity and the identification of novel therapeutic ribozymes.

In 1998, Dr Burgin became faculty at San Diego State University where he directed an active research lab studying the mechanism of DNA recombinases, topoisomerases, and phosphodiesterases. In 2001, Dr Burgin joined deCODE biostructures as Director of Molecular Biology and remains Adjunct Associate Professor at SDSU. He currently oversees gene synthesis, cloning, bacterial fermentation and assay development at deCODE.

Launch Presentation