DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

121. Protein Expression in Methanococccus jannaschii and Pyrococcus furiosus

C.S. Giometti¹, S.L. Tollaksen¹, H. Lim², J. Yates², J. Holden³, A. Lal Menon³, G. Schut³, M.W.W. Adams³, C. Reich⁴, and G. Olsen⁴

¹Argonne National Laboratory, Argonne, IL; ²University of Washington, Seattle, WA; ³University of Georgia, Athens, GA; and ⁴University of Illinois, Urbana, IL

csgiometti@anl.gov

Complete genome sequences are now available for both Methanococcus jannaschii and Pyrococcus furiosus. The open reading frame (ORF) sequences from these completed genomes can be used to predict the proteins synthesized, but laboratory methods are needed to verify those predictions. Two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry of peptides isolated from the gels is being used to determine the constitutive expression of proteins from these two Archaea and to explore the regulation of expression of non-constitutive proteins. The most abundant proteins (i.e., those easily detectable by staining with Coomassie Blue R250) from cells grown in minimal nutrient media have been isolated and analyzed. Using a combination of matrix-assisted laser desorption ionization (MALDI) and tandem mass spectrometry, 100 proteins expressed by M. jannaschii and 50 proteins expressed by P. furiosus have been related to specific ORFs in the respective genome sequences. The molecular weights and isoelectric points determined by the positions of proteins in the 2DE patterns are compared with the ORF-predicted molecular weights and isoelectric points for each microbe. Numerous instances of multiple proteins with different molecular weights or isoelectric points being associated with the same ORF have been observed. Possible reasons for such multiplicity include the incomplete unfolding of these highly stable proteins prior to electrophoresis, the non-dissociation of subunits, post-translational modifications such as phosphorylation (multiple proteins with the same identity but different isoelectric points) or peptide cleavage (multiple proteins with the same identity but different molecular weights). Preliminary experiments to change the protein expression of these organisms by altering growth conditions have revealed significant quantitative changes in a small number of the proteins visible in 2DE patterns. Correlation of proteins expressed with specific ORFs is now focused on those proteins showing quantitative changes in expression and on less abundant proteins. The observed protein abundances and changes in abundance from these proteomic studies could be useful for validation of predictions of protein expression based on ORFs.

This work is supported under Contract No.W-31-109-ENG-38 with the U.S. Department of Energy.