Crystallisation of a modified fibrinogen

Polymer‐driven crystallization

“…Proteins with a high crystallization propensity crystallize in many conditions (Segelke 2001), whereas some recalcitrant proteins appear to require modifications to make them crystallizable (D'Arcy 1994; Dale et al 2003; Derewenda 2004a; Keenan et al 2005). Several methods are available to increase crystallization propensity, including limited proteolysis to remove disordered regions (Tooney and Cohen 1972; Cohen and Tooney 1974; Kwong et al 1999), mutation of residues on the protein's surface that might be involved in crystal contacts (Lawson et al 1991; D'Arcy et al 1999; Czepas et al 2004; Derewenda 2004a,b; Janda et al 2004; Derewenda and Vekilov 2006), deglycosylation to diminish heterogeneity (Kwong et al 1999), and antibody co‐crystallization to rigidify the target and add potential crystal contacts (Iwata et al 1995; Kleymann et al 1995; Ostermeier et al 1995; Ostermeier and Michel 1997; Hunte et al 2000; Zhou et al 2001; Hunte and Michel 2002; Jiang et al 2003; Lee et al 2005). Although useful, these methods must be applied in an ad hoc fashion for each target.…”

A Model for Fibrinogen: Domains and Sequence

“…JOHN The early trinodular Hall-Slayter model for fibrinogen (1), based on electron microscope images of shadowed molecules, has provided a valid, simplified picture of the molecule despite a variety of other confficting proposals (2). Detailed information on the morphology and dimensions of fibrinogen has been derived from analysis of crystalline arrays (3)(4)(5)(6)(7)(8)(9). Native fibrinogen does not crystallize, but crystals have been produced after limited cleavage of the molecule by a bacterial protease.…”

“…1 Fig. 3) and 30-A x-ray data from P21 crystals (4)(5)(6)(7)(8). Refinement offibrinogen models against the x-ray data to 30 A (105 unique reflections) was carred out by an iterative process with the use of constraints from the image analysis of electron micrographs of the P2, crystal.…”

“…Cleavage in these flexible single chain regions by the enzyme from Pseudomonas and a variety of other enzymes (4,6,8) appears at present to be a necessary step in crystallizing fibrinogen.…”

“…5 also depicts the a domain formed by the COOH-termini of the Aa chains folding back to interact at the center of the molecule as would be seen in native fibrinogen. SV40 and polyoma virus enhancers (4,5). Enhancers are cis-acting promoter elements that are required for efficient transcription of some viral and cellular genes and can also activate transcription from heterologous promoters.…”