Gene to crystal flowchart. Gene synthesis can be integrated into an experimental pipeline where proteins can be designed for crystallization screening. It is possible to design variations of protein constructs by exploiting the SeqTBIO method of sequential DNA assembly (path 1 to 6). Different constructs can be screened for expression in 96-well format array and evaluated for the protein solubility and stability (path 7). In our case, we used heat stability as a biochemical selection criterion. However, recombinant tags can be easily incorporated at the DNA synthesis level whereby the resulting expressed proteins can be selected with affinity binding matrices (e.g. His or GST tags). Large scale production can then be performed for crystallization trials (path 8–9). Initial crystals obtained would undergo optimization and preliminary X-ray analysis (path 10). Further protein sequence changes can be made if necessary for further processing (path 11).
Marsic et al. BMC Biotechnology 2008 8:44 doi:10.1186/1472-6750-8-44