Protein encoding open reading fromes(ORFs) were obtoined from public ORF librories or independently synthesized by the lobs of HengZhu & Seth Backshaw at Johns Hopkins University. Using Gateway cloningin E. coli, individual ORF DNA sequences were shuttled into a yeast high-copy expression vector (pEGH-A) that produces N-terminal GST-fusion proteins. Protein-encoding vectors were rescued from E.coli and used to transform S.cerevisiae yeast. Yeast were grown on plates to select individual transformed clones. Successfully transformed yeast clones were bideirectionally sequenced to confirm correct ORF insertion and expected protein identity.Clones failing sequence QC were resynthesized. To produce proteins, sequence-confirmed yeast clones are grown,lysed,and harvested.0.1% Triton buffers remove lipids. GST-fusion proteins are purified via GST-binding agarose beads. For each protein production lot,random wells are run on gels to confirm purity and expected molecular weight. Plates with a protein sample failing gel QC remade. The prolein production lot is aliquoted and frozen. Each protein lot can produce around 14000 HuProt TM arrays. Arravs are printed in batches of 600 on nitrocellulose sides using Arrayjet printers. Two slides from each batch are stained with anti-GST; >95% of spots must be GST+ for the array batch to pass QC. This confirms round-trip success of GST-fusion libranry synthesis & printing.
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