ACM SIGGRAPH International Conference on Virtual Reality Continuum and Its Applications in Industry Nanyang Technological University, Singapore, June 16-18, 2004. Method. Reference. Rationale.
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ACM SIGGRAPH International Conference on
Virtual Reality Continuum and Its Applications in Industry
Nanyang Technological University, Singapore, June 16-18, 2004
The bottleneck in structural genomics projects is over expression of the target proteins in soluble form. Large-scale genome sequencing has produced a huge amount of information on gene sequence. Large-scale analysis of gene product has been hampered by the inability to produce sufficient quantities of protein in soluble form [Stam 2001]. By the application of directed evolution technique and prepared soluble mutants of the Mycobacterium Tuberculosis Rv2002 gene product, which had been expressed as inclusion bodies in the E-coli.
Crystal Structure of Rv2002 Gene Product from Mycobacterium Tuberculosis (1NNF)
Seow Joo Hong
Fig 1. Primary Structure
Fig 2. Secondary Structure
Fig 4. Ligands
Fig 3. Space filling
It is possible to perform whole scale gene expression in E-coli where the expressed proteins may not fold properly and may accumulate as inclusion bodies. A conventional way to overcome the solubility difficulty is site-directed mutagenesis of one or a few amino acids which will be slow and extensive effort required. A new directed evolution strategy which the gene encoding the protein is subjected to random mutations and the soluble mutants are selected by the coding GFP ( Green Fluorescent Protein). Figures 1-4 illustrates the structure of the proteins. The target protein is fused to N-terminus of the GFP. The GFP folds properly and emits green light only when the upstream target mutant protein folds properly (Fig 5).
Fig 5. Target Mutant
Rv2002 a reduction enzyme in mycobacterium tuberculosis was used as an example. Random mutagenesis and selections rendered a dramatic improvement in the molecule’s solubility when expressed in E-coli.
STAM, J. 2001. A Simple Fluid Solver Based on the FFT. Journal of Graphics Tools 6, 2, 43–52.