The CELL-FREE PROTEIN PRODUCTION Team was headed by John Markley, PhD, and was responsible for high-throughput protein production for structural investigations from a wheat germ cell-free expression system.
One of the major bottlenecks in high-throughput structure determination effort is protein expression. Structural proteomics efforts will require expression and purification of thousands of proteins and/or protein fragments. The ability to obtain labeled proteins is an essential aspect required for rapid progress in structural determinations. Critical examples are:
However, many individual proteins cannot be expressed in soluble form in bacteria and are, therefore, not suitable for E. coli cell-based production methodologies. Insolubility arises either from an intrinsic property of a protein (for example, aggregation due to a very hydrophobic patch on the surface) or because the protein is not susceptible to the folding mechanisms in the expression host; in which case there is an aggregation of folding intermediates. These include one-third to one half of prokaryote proteins. This proportion is likely to be higher for eukaryotic proteins, particularly those that comprise multiple domains, those that require cofactors or protein partners for proper folding, or those that require extensive post-translational modification. The development of new systems and strategies capable of synthesizing any desired soluble, labeled protein, or protein fragment on a preparative scale as alternatives E. coli cell-based production is one of the most important tasks in biotechnology today.