In order to elucidate various biological phenomena occurring in vivo, it is essential to determine the structure of proteins. This article will focus on expression of proteins for X-ray analysis.
For performing structure analysis, the first challenge is to establish an expression system. Structure analysis requires large amounts of proteins with high purity, so it is essential to establish a large-scale protein expression and purification systems. Even target proteins that are only present in trace amounts in vivo can be overexpressed as recombinant proteins using genetic engineering techniques and appropriate hosts. In such cases, it is crucial to select an appropriate expression system from numerous existing protein expression systems. Some expression systems cannot be used or are more difficult to use, depending on whether the protein of interest is of prokaryotic or eukaryotic origin. For multi-domain proteins or proteins expected to undergo large structure change, regions containing only certain domains can be selectively expressed to suppress structure change. For rapid purification of the expressed proteins, in most cases, purification tags are generally added to the N- and/or C-terminals of proteins. Sometimes, the expressed proteins do not fold properly and are expressed as “inclusion bodies”, which cannot be directly used as starting materials for purification. As can be seen, there are many challenges to be overcome to obtain proteins that can be used as starting materials for structure analysis.