Design of high-throughput methods of protein production for structural biology.

Introduction High-throughput protein expression and purification has a central, pivotal role in structural genomics. In fact, crystallographic-quality protein production on the scale required to generate tens to hundreds of different proteins per day will probably be the greatest obstacle for the conversion of protein structure determination to a high-throughput format. High-throughput efforts in structural biology place unique restrictions on protein expression and purification. First, the majority of gene constructs must be expressed in a synchronous fashion. Second, the purification protocol applied to the majority of expressed proteins must be as similar as possible and produce the very high-quality material that is needed for structural studies. The first requirement can be addressed by employing either large or small N-terminal expression tags, and the second hurdle can be circumvented with the use of affinity purification tags. However, the drawback in incorporating affinity tags in crystallization studies is that in many cases these tags introduce flexible portions to the protein of interest that are not conducive to crystallization or lead to various forms of microheterogeneity. Protease cleavage sites allow removal of these flexible tag regions, but conditions often have to be optimized for each reaction, requiring fine-tuned processing to be incorporated in a high-throughput environment. Given the above caveats on current technology, however, affinity-tag systems are still the most useful to date given the restrictions placed on high-throughput methods.