The number of published 3D structures has increased exponentially in the last decade and the resulting mass of structural data has contributed significantly to the understanding of mechanisms underlying the biology of living cells. However, these mechanisms are so complex that structural biologists face still greater challenges, such as the study of higher-order functional complexes. As an example, we can mention the protein complexes that assemble around activated growth factor receptors to allow the transduction of extracellular signals through the membrane and inside the cell (1). Because of their diverse intrinsic properties, proteins exhibit variable difficulty for structural biology studies. Before the rise of recombinant expression methods, only a minority of protein structures were determined, representing mainly favourable cases: proteins of high abundance in their natural source which could be purified and crystallized, in contrast to rare proteins that were often refractory to crystallization. The advent of methods for recombinant protein overexpression was a breakthrough in this area. It was followed by an increasing number of publications describing the crystallization of proteins, not under their native form, but in modified versions after sequence engineering. First we will consider the classical use of molecular biology applied to optimize the expression system for a recombinant protein for structural biology, without modification of its sequence. In the second part, we will deal with molecular biology procedures aimed at engineering the properties of a protein through sequence modifications in order to make its crystallization possible. In the last part we will give an example where molecular biology can help solve a crystallographic problem, namely that of phase determination by introducing anomalous scatterers (e.g. selenium atoms) into the protein of interest. Whenever extraction of a protein from its natural source appears unsuitable for structural studies, molecular biology resources can be brought in, initially aiming at choosing and setting up an appropriate expression system. This initial approach could involve comparing various expression hosts and vectors and deciding if the protein is to be produced as a fusion to facilitate its purification.
A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells