Abstract
We describe a straightforward crystallogenesis protocol leading to the preparation of protein crystals suitable for structure determination that involves protein expression and purification, refolding of the overexpressed protein, search of optimal crystallization conditions and diffraction data collection on native and selenomethionine substituted-crystals. The protocol is exemplified with epididymal-specific lipocalin (rLcn6), a newly discovered monomeric protein of 19 kDa that may play an important role in sperm maturation. This protein was cloned from Norway rat (Rattus norvegicus) genome and expressed as insoluble inclusion bodies in Escherichia coli. After refolding of the purified protein, microcrystals were obtained after sparse matrix screening. Optimization of conditions after stepwise incremental initial conditions led to single crystals belonging to space group P2(1)2(1)2(1) and data set at 1.90 angstrom. After attempts with several models, initial phasing was not found by molecular replacement. A similar methodological scheme was used to grow quality crystals of the selenomethionine-substituted rLcn6 protein and collect diffraction data at 2.0 angstrom, allowing phasing and structure resolution. This protocol may be of particular help when overproduction results in denatured proteins within inclusion bodies, a situation that often occurs especially with proteins from eukaryotes, as well as with structural genomic projects.