title: JHMI/Zhu_YEAST_14K_v1.0
status: Public on Dec 05 2008
contact.city: Baltimore
contact.first: Eitan
contact.last: Halper-Stromberg
contact.email: ehalper2@jhmi.edu
contact.state: MD
contact.address: 733 N Broadway
contact.country: USA
contact.institute: Johns Hopkins
organism: Saccharomyces cerevisiae
manufacture_protocol: We have constructed a yeast proteome microarray containing approximately 80% yeast proteins and screened it for a number of biochemical activities. We first built a high-quality collection of 5800 yeast open reading frames (ORFs) (93.5% of the total) cloned into a yeast high-copy expression vector using recombination cloning. The yeast proteins are fused to glutathione S-transferase-polyhistidine (GST-HisX6) at their NH2-termini and expressed in yeast using the inducible GAL1 promoter. The yeast expression strains contain individual plasmids in which the correct yeast ORFs have been shown to be fused in-frame to GST by DNA sequencing. The proteins were expressed in yeast to help ensure that the proteins were modified and folded properly. Using a 96-well format, 1152 samples were purified at once from yeast extracts using glutathione-agarose beads. We included 0.1% Triton in the lysis buffer and washes to ensure that the purified proteins were free of lipids. The quality and quantity of the purified proteins were monitored using immunoblot analysis of 60 random samples. Greater than 80% of the strains produced detectable amounts of fusion proteins of the expected molecular weight.
  To prepare the proteome chips, we printed 6566 protein preparations, representing 5800 different yeast proteins, in duplicate onto glass slides using a commercially available microarrayer. Our initial experiments used aldehyde-treated microscope slides in which fusion proteins attach to the surface through primary amines at their NH2-termini or other residues of the protein. In subsequent experiments, we spotted proteins onto nickel-coated slides, in which the fusion proteins attach through their HisX6 tags and presumably uniformly orient away from the surface. Although both slides were successful, the nickel-coated slides gave superior signals for our particular protein preparations.
  To determine how much fusion protein was covalently attached to different glass surfaces and the reproducibility of the protein attachment, we probed the chips with antibodies to GST (anti-GST). More than 93.5% of the protein samples gave signals significantly above background (i.e., greater than 10 fg of protein), and 90% of the spots contained 10 to 950 fg of protein. Our results also demonstrate that it is feasible to spot, with excellent resolution, 13,000 protein samples in one-half the area of a standard microscope slide. To test the reproducibility of the protein spotting, we compared the signals from each pair of duplicated spots with one another; 95% of the signals were within 5% of the average.