The polyethylene glycol (PEG) positive effect in protein crystallization

We have used two general approaches to facilit:1te the growth of protein c1ystals for X-ray diffiaction experiments.The first approach is called SAF, for smallest, active fi:agment.SAF is simply a logical extension of the old observation that proteolytic fiagments of proteins often both harbour complete biological activity and form useful c1ystals more reaclily than the full length protein.However, rather than depend on the fortuitous position of protease recognition sites, SAF couples partial proteolysis with deletion analysis in order to refine the bmmdaries of the domain of interest.In practice, an iterative cycle of partial proteolysis, deletion analysis, biochemical assays, protein over-expression and c1ystallization is used to identify a domain that fonm diffraction-quality crystals.SAF capitalizes on the ease and rapidity of subcloning, overexpressing and pmifying proteins as well as the technical ease and availability ofN-tenninal sequencing and mass spectrometry.We have used the SAF approach for three proteins, and we have generated fragments of each that were suitable for str11cture detemrination.The second approach.lipid-layer seeding, uses lipid monolayers to seed c1ystal growth.We observed several years ago that two-din1ensional protein cryst:1ls grown on lipid layers effectively nucleate the epita\.ialgrowth of three din1ensional protein c1yst:1ls.In this way, crystals suitable for X -ray crystallography can be grow11 more rapidly, and at subst:mtially lower protein concentrations and precipit:mts than for conventional c1ystal 1:Iials.The methodology has now been developed such that the procedure takes only a few seconds per c1ystal trial.We are now applying the method to an assortment of proteins in an effort to demonstrate the generality of the approach.