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 indication of the purity of the preparation with respect to nucleic acids. We show here that for low-molecular-weight recombinant proteins purified from Escherichia coli, a low A(280)/A(260) ratio can also result from contamination with UDP-linked murein precursors derived from bacterial cell wall metabolism. Although these precursors are small molecules of molecular weight 1000-1200, they comigrate in gel filtration with recombinant human FKBP (MW 11,820). This gel filtration behavior, which is distinct from that of unmodified mononucleotides, does not reflect binding interactions with FKBP, but is an intrinsic property of these precursors. Therefore, these molecules would be expected to copurify with other low-molecular-weight proteins, especially in the abbreviated purification protocols made possible by freeze-thaw release of recombinant proteins from E. coli (Johnson, B. H., and Hecht, M. H. (1994) BioTechnology 12, 1357-1360). Several alternative strategies are discussed for integrating these findings into the design of improved purification procedures for low-molecular-weight recombinant proteins.
Ram
M K
MK
ARIAD Pharmaceuticals Inc., 26 Landsdowne Street, Cambridge, Massachusetts, 02139, USA.
Andrade
L J
LJ
Phillips
T B
TB
van Schravendijk
M R
MR
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry*; D012116:Resins, Plant; D013056:Spectrophotometry, Ultraviolet; D014530:Uridine Diphosphate / Q000737:chemistry / Q000378:metabolism*
D010457:Peptidoglycan; D011994:Recombinant Proteins; D012116:Resins, Plant; D014530:Uridine Diphosphate