Protein Expression Labs
A major bottleneck in many biophysical methods and determining structures using X-ray crystallography lies in expression of sufficient quantities of highly pure protein. These web pages have been developed by the Crystallography Group (aka 'the Structural Biology' group) and describe methodology that has been used to successfully express and purify a variety of proteins from human and prokaryotic genes. Every protein is different and a protein expression strategy is required to tackle each protein expression project. We have developed a multi-tiered platform of protein expression involving an array of vectors and hosts. Special expertise is required for secreted proteins such as cellular receptors. These web pages describe several vector and host systems that can be combined with different tags and used for expression and purification.
1. E.Coli Vectors and Hosts
2. Insect and Mammalian Cell Vectors and Hosts
Domain Boundary Definition
Accurate domain boundaries can be critical for obtaining high levels of protein expression as loose ends at the N and C-termini can promote aggregation and these are also highly inhibitory to protein crystallisation. Two methods are employed
1. Bioinformatics
Bioinformatics servers’ phyre and domain fishing are useful for determining boundaries by examining fold libraries;
http://www.sbg.bio.ic.ac.uk/~phyre/
Bennett-Lovsey RM, Herbert AD, Sternberg MJE, Kelley LA. Proteins: Structure, Function, Bioinformatics, vol 70(3) 611-625 (2008).
http://www.bmm.icnet.uk/~3djigsaw/dom_fish/
Contreras-Moreira,B. and Bates,P.A. (2002). Domain Fishing: a first step in protein comparative modelling. Bioinformatics 18: 1141-1142
Regions with inherent disorder can be predicted using the RONN server
http://www.strubi.ox.ac.uk/RONN
2. Biochemical: Limited proteolysis
Once you have purified soluble protein proteolysis can be used to define domain boundaries. A panel of proteases is used to clip off loose ends. These include amongst others trypsin, chymotrypsin, endoprotease Lys-C, subtilisin, elastase and bromelain which have a wide range of specificities. The method is described in this review by Simon Hubbard.
The structural aspects of limited proteolysis of native proteins.
Hubbard SJ. Biochim Biophys Acta. 1998 Feb 17;1382(2):191-206. Review.