Production and Characterization of Recombinant Proteins for NMR Structural Studies

Abstract

The recent rapid development of multidimensional and multinuclear methodology in NMR has steadily extended the range of proteins whose three-dimensional structures can be fully described by this technique alone. We review here some of the expression and labeling strategies used to produce recombinant proteins, protein domains, and subdomains, for this purpose. In planning expression and isolation strategies for such materials, there are several basic requirements for the end product that must be taken into consideration at the outset:

• 1. Homogeneity: For NMR experiments, at least 95% of the polypeptide product should ideally be a single molecular entity. The significance of impurities depends on their origin and molecular weight. Ten percent of a l00-kDa impurity in a 15-kDa protein would usually be of little consequence because of low signal intensity. However, low-molecular weight impurities carried through purification schemes can often obscure regions of the spectrum even when present at low concentrations. Similarly, microheterogeneity in the amino acid sequence owing to processing at the chain termini or (less commonly) mutation or alternative splicing should be avoided if possible.

• 2. Solubility: Successful structural studies usually require samples of between 0.5 and 2.5 mM, so the protein must be soluble at these concentrations without aggregation. Even weak association processes can result in a significant loss of spectral information. Solubility can be manipulated within limits by formulation conditions (see Section 3.) but is the major reason (apart from the size of the protein itself) for re-engineering and fragmentation approaches.

• 3. Quantity. A full set of experiments to complete sequential assignments and acquisition of nOe datasets will usually require 1–5 µmol of pure protein.