Purification and crystallization of a proteolytic fragment of Escherichia coli pyruvate formate-lyase

Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyas e (PFL) is the first reaction after the production of pyruvate in the glyco lytic pathway. Crystallization trials with Escherichia coli PFZ were unsucc essful and therefore limited proteolysis was used to produce a stable cryst allizable N-terminal protein fragment by trypsin cleavage. The molecular we ight 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 recom binant protein fragment was crystallized by sitting-drop vapour diffusion u sing 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 gr oup P6(1) or P6(5) with cell dimensions a = b = 140.4, c = 215.3 Angstrom a nd two molecules per asymmetric unit. X-ray diffraction data were collected from 20 to 3.2 Angstrom resolution from a single frozen crystal on a synch rotron-radiation beamline.