title: Robust microarray production of freshly expressed proteins in a human milieu.
abstract: OBJECTIVE
  In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT (HCIVT)) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu.
  
  
  METHODS
  Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by HCIVT compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine.
  
  
  RESULTS
  HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than ten times higher than RRL, in both Western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array.
  
  
  CONCLUSIONS
  HCIVT is a robust in vitro transcription and translation system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.
authors.affiliation: Virginia G. Piper Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ 85287-6401, USA.
authors.forename: Fernanda
authors.initials: F
authors.lastname: Festa
authors.forename: Sean M
authors.initials: SM
authors.lastname: Rollins
authors.forename: Krishna
authors.initials: K
authors.lastname: Vattem
authors.forename: Margarita
authors.initials: M
authors.lastname: Hathaway
authors.forename: Phillip
authors.initials: P
authors.lastname: Lorenz
authors.forename: Eliseo A
authors.initials: EA
authors.lastname: Mendoza
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Yu
authors.forename: Ji
authors.initials: J
authors.lastname: Qiu
authors.forename: Greg
authors.initials: G
authors.lastname: Kilmer
authors.forename: Penny
authors.initials: P
authors.lastname: Jensen
authors.forename: Brian
authors.initials: B
authors.lastname: Webb
authors.forename: Ed T
authors.initials: ET
authors.lastname: Ryan
authors.forename: Joshua
authors.initials: J
authors.lastname: LaBaer
mesh_terms: D000818:Animals; D015153:Blotting, Western; D006801:Humans; D040081:Protein Array Analysis; D014176:Protein Biosynthesis; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
chemical_list: D011506:Proteins