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

Veli Matti Leppänen, Camran V. Parast, Kenny K. Wong, John W. Kozarich, Adrian Goldman

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 PB5 with cell dimensions a = b = 140.4, c = 215.3 Å and two molecules per asymmetric unit. X-ray diffraction data were collected from 20 to 3.2 Å resolution from a single frozen crystal on a synchrotron-radiation beamline.

Original languageEnglish
Pages (from-to)531-533
Number of pages3
JournalActa Crystallographica Section D: Biological Crystallography
Volume55
Issue number2
DOIs
StatePublished - Feb 1999

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