ABSTRACT
Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways. sam1(-) sam2(-) mutants, in which the conversion of methionine to S-adenosylmethionine is blocked, exhibit reduced selenomethionine toxicity compared with wild-type yeast, increased production of protein during growth in selenomethionine, and efficient replacement of methionine by selenomethionine, based on quantitative mass spectrometry and x-ray crystallography. The structure of yeast tryptophanyl-tRNA synthetase was solved to 1.8 A by using multiwavelength anomalous dispersion phasing with protein that was expressed and purified from the sam1(-) sam2(-) strain grown in selenomethionine. Six of eight selenium residues were located in the structure.
Subject(s)
S-Adenosylmethionine/antagonists & inhibitors , S-Adenosylmethionine/biosynthesis , Saccharomyces cerevisiae/metabolism , Selenomethionine/pharmacokinetics , Crystallography, X-Ray , S-Adenosylmethionine/chemistry , Saccharomyces cerevisiae/chemistry , Selenomethionine/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We developed a method to co-express protein pairs from collections of otherwise identical Escherichia coli plasmids expressing different ORFs by incorporating a 61-nucleotide sequence (LINK) into the plasmid to allow generation of tandem plasmids. Tandem plasmids are formed in a ligation-independent manner, propagate efficiently, and produce protein pairs in high quantities. This greatly facilitates co-expression for structural genomics projects that produce thousands of clones bearing identical origins and antibiotic markers.
