PMID:10089368

Citation	Leppänen, VM, Parast, CV, Wong, KK, Kozarich, JW and Goldman, A (1999) Purification and crystallization of a proteolytic fragment of Escherichia coli pyruvate formate-lyase. Acta Crystallogr. D Biol. Crystallogr. 55:531-3
Abstract	Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyase (PFL) is the first reaction after the production of pyruvate in the glycolytic pathway. Crystallization trials with Escherichia coli PFL were unsuccessful and therefore limited proteolysis was used to produce a stable crystallizable N--terminal protein fragment by trypsin cleavage. The molecular weight of this cleavage product was found to be 69.6 kDa by MALDI MS analysis, and the DNA sequence corresponding to this fragment was cloned. The recombinant protein fragment was crystallized by sitting-drop vapour diffusion using polyethylene glycol 1000 as precipitant. The crystals, which grew to 2 mm in length and 0.2 mm in cross section, belong to the hexagonal space group P61 or P65 with cell dimensions a = b = 140.4, c = 215.3 A and two molecules per asymmetric unit. X--ray diffraction data were collected from 20 to 3.2 A resolution from a single frozen crystal on a synchrotron-radiation beamline.
Links	PubMed
Keywords	Acetyltransferases/chemistry; Base Sequence; Crystallization; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli/genetics; Hydrolysis; Peptide Fragments/chemistry; Peptide Fragments/isolation & purification; Protein Conformation; Recombinant Proteins/chemistry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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