Identification of UDP-linked murein precursors as contaminants in recombinant proteins of low molecular weight

The A(280)/A(260) ratio of a purified protein is frequently used as an indi cation of the purity of the preparation with respect to nucleic acids. We s how here that for low-molecular-weight recombinant proteins purified from E scherichia coli, a low A(280)/A(260) ratio can also result from contaminati on with UDP-linked murein precursors derived from bacterial cell wall metab olism. Although these precursors are small molecules of molecular weight 10 00-1200, they comigrate in gel filtration with recombinant human FKBP (MW 1 1,820). This gel filtration behavior, which is distinct from that of unmodi fied mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules wo uld be expected to copurify with other low-molecular-weight proteins, espec ially in the abbreviated purification protocols made possible by freeze-tha w release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies a re discussed for integrating these findings into the design of improved pur ification procedures for low-molecular-weight recombinant proteins. (C) 199 9 Academic Press.