Document Detail


Robust microarray production of freshly expressed proteins in a human milieu.
MedLine Citation:
PMID:  23027544     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
PURPOSE: 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.
EXPERIMENTAL DESIGN: 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.
CONCLUSION AND CLINICAL RELEVANCE: 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:
Fernanda Festa; Sean M Rollins; Krishna Vattem; Margarita Hathaway; Phillip Lorenz; Eliseo A Mendoza; Xiaobo Yu; Ji Qiu; Greg Kilmer; Penny Jensen; Brian Webb; Ed T Ryan; Joshua LaBaer
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-05-17
Journal Detail:
Title:  Proteomics. Clinical applications     Volume:  7     ISSN:  1862-8354     ISO Abbreviation:  Proteomics Clin Appl     Publication Date:  2013 Jun 
Date Detail:
Created Date:  2013-06-19     Completed Date:  2013-11-27     Revised Date:  2014-03-20    
Medline Journal Info:
Nlm Unique ID:  101298608     Medline TA:  Proteomics Clin Appl     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  372-7     Citation Subset:  IM    
Copyright Information:
© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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MeSH Terms
Descriptor/Qualifier:
Animals
Blotting, Western
Humans
Protein Array Analysis*
Protein Biosynthesis
Proteins / analysis,  metabolism*
Rabbits
Reticulocytes / metabolism
Grant Support
ID/Acronym/Agency:
U01 AI077883/AI/NIAID NIH HHS; U01AI077883/AI/NIAID NIH HHS; U01CA117374/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Proteins

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