title: JHUHIT Human 17K v2009
status: Public on Oct 25 2011
contact.city: Baltimore
contact.first: Mary Katherine
contact.last: Tarrant
contact.email: marykatherine.tarrant@jhu.edu
contact.state: MD
contact.address: 725 N. Wolfe Street, 317 Hunterian Bldg.
contact.department: Pharmacology and Molecular Sciences
contact.country: USA
contact.institute: Johns Hopkins University School of Medicine
contact.phone: 410-614-0322
organism: Homo sapiens
description: Human proteins expressed and purified from yeast
manufacture_protocol: DNA corresponding to the human proteins (ORFs from the Invitrogen collection) was cloned into the pEGH-A vector (a derivative of the yeast glutathione S-transferase, GST, vector) using the Gateway system.  The plasmid DNAs corresponding to the GST-fusion proteins were transformed into yeast (WT Y258A) for protein expression and purification.  Cultures were grown at 30° to an optical density at 600 nm of 0.7 to 0.9 and induced with 2% galactose for 4 to 6 h.  Harvested cells were lysed with zirconia beads in lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride plus Roche protease inhibitor cocktail).  GST fusion proteins were bound to glutathione beads for 1 h at 4°C and washed three times with wash I buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and washed three times with wash II buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 0.1% 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride) and eluted in elutions buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 30% glycerol, 0.03% Triton X-100, 0.1% 2-mercaptoethanol, 40 mM glutathione).  The eluate was collected through a filter unit and stored in plates in a -80°C freezer.  The protein products were printed in duplicate on modified glass slides (Full Moon, Biosystems) using a NanoPrint Microarrayer (ArrayIt).  To reduce the merged features, printing is done at lower humidity (~30%) using SMP3 pins from ArrayIt with column and row spacing 135 microns and 170 microns, respectively.