Robust microarray production of freshly expressed proteins in a human milieu

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

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)372-377
Number of pages6
JournalProteomics - Clinical Applications
Volume7
Issue number5-6
DOIs
StatePublished - Jun 2013

Fingerprint

Microarrays
Proteins
Protein Array Analysis
Reticulocytes
Transcription
Rabbits
Western Blotting
Protein folding
Biomarkers
Pathogens
Protein Folding
Design of experiments
Proteomics
Assays
Vaccines
Cells
Research Design
Antigens

Keywords

  • Human Coupled IVT
  • In vitro transcription and translation
  • NAPPA
  • Protein microarray

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Robust microarray production of freshly expressed proteins in a human milieu. / Festa, Fernanda; Rollins, Sean M.; Vattem, Krishna; Hathaway, Margarita; Lorenz, Phillip; Mendoza, Eliseo A.; Yu, Xiaobo; Qiu, Ji; Kilmer, Greg; Jensen, Penny; Webb, Brian; Ryan, Ed T.; LaBaer, Joshua.

In: Proteomics - Clinical Applications, Vol. 7, No. 5-6, 06.2013, p. 372-377.

Research output: Contribution to journalArticle

Festa, F, Rollins, SM, Vattem, K, Hathaway, M, Lorenz, P, Mendoza, EA, Yu, X, Qiu, J, Kilmer, G, Jensen, P, Webb, B, Ryan, ET & LaBaer, J 2013, 'Robust microarray production of freshly expressed proteins in a human milieu', Proteomics - Clinical Applications, vol. 7, no. 5-6, pp. 372-377. https://doi.org/10.1002/prca.201200063
Festa F, Rollins SM, Vattem K, Hathaway M, Lorenz P, Mendoza EA et al. Robust microarray production of freshly expressed proteins in a human milieu. Proteomics - Clinical Applications. 2013 Jun;7(5-6):372-377. https://doi.org/10.1002/prca.201200063
Festa, Fernanda ; Rollins, Sean M. ; Vattem, Krishna ; Hathaway, Margarita ; Lorenz, Phillip ; Mendoza, Eliseo A. ; Yu, Xiaobo ; Qiu, Ji ; Kilmer, Greg ; Jensen, Penny ; Webb, Brian ; Ryan, Ed T. ; LaBaer, Joshua. / Robust microarray production of freshly expressed proteins in a human milieu. In: Proteomics - Clinical Applications. 2013 ; Vol. 7, No. 5-6. pp. 372-377.
@article{c130fe05e5d0438580f54a8b43ca170d,
title = "Robust microarray production of freshly expressed proteins in a human milieu",
abstract = "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.",
keywords = "Human Coupled IVT, In vitro transcription and translation, NAPPA, Protein microarray",
author = "Fernanda Festa and Rollins, {Sean M.} and Krishna Vattem and Margarita Hathaway and Phillip Lorenz and Mendoza, {Eliseo A.} and Xiaobo Yu and Ji Qiu and Greg Kilmer and Penny Jensen and Brian Webb and Ryan, {Ed T.} and Joshua LaBaer",
year = "2013",
month = "6",
doi = "10.1002/prca.201200063",
language = "English (US)",
volume = "7",
pages = "372--377",
journal = "Proteomics - Clinical Applications",
issn = "1862-8346",
publisher = "Wiley-VCH Verlag",
number = "5-6",

}

TY - JOUR

T1 - Robust microarray production of freshly expressed proteins in a human milieu

AU - Festa, Fernanda

AU - Rollins, Sean M.

AU - Vattem, Krishna

AU - Hathaway, Margarita

AU - Lorenz, Phillip

AU - Mendoza, Eliseo A.

AU - Yu, Xiaobo

AU - Qiu, Ji

AU - Kilmer, Greg

AU - Jensen, Penny

AU - Webb, Brian

AU - Ryan, Ed T.

AU - LaBaer, Joshua

PY - 2013/6

Y1 - 2013/6

N2 - 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.

AB - 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.

KW - Human Coupled IVT

KW - In vitro transcription and translation

KW - NAPPA

KW - Protein microarray

UR - http://www.scopus.com/inward/record.url?scp=84876461875&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84876461875&partnerID=8YFLogxK

U2 - 10.1002/prca.201200063

DO - 10.1002/prca.201200063

M3 - Article

C2 - 23027544

AN - SCOPUS:84876461875

VL - 7

SP - 372

EP - 377

JO - Proteomics - Clinical Applications

JF - Proteomics - Clinical Applications

SN - 1862-8346

IS - 5-6

ER -