Abstract
In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, we established a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP was based on a transient expression system in HEK293-EBNA cells at 100 to 500 mL culture scale, combined with an automated affinity purification procedure targeting > 75% purity. This was followed by a semiautomated protein sample logistics to provide biologists with quality-controlled and 96 well formatted protein aliquots amenable to cell-based assays. Over a 4-year period, beginning in 2001, we performed over 7,500 transfections representing over 2,200 registered proteins, including both novel and reference proteins, at an average production of 280 proteins/ month with a peak production of 320 proteins/month. All these proteins have been tested in more than 50 different cell-based assays. This article describes the major process steps and highlights the optimization required to maintain novel protein production while supporting both stock replenishment and scale-up productions.
Keywords: Protein, high-throughput screening, secretome, expression, purification, mammalian, 96-well, HEK293 ells
Combinatorial Chemistry & High Throughput Screening
Title: A High-Throughput Mammalian Protein Expression, Purification, Aliquoting and Storage Pipeline to Assemble a Library of the Human Secretome
Volume: 9 Issue: 9
Author(s): Thierry Battle, Bruno Antonsson, Georg Feger and Dominique Besson
Affiliation:
Keywords: Protein, high-throughput screening, secretome, expression, purification, mammalian, 96-well, HEK293 ells
Abstract: In the post-human genome-sequencing era, the availability of recombinant proteins has become crucial for the identification of proteins with therapeutic potential. Based upon bioinformatic coding predictions of the genes for putative secreted proteins, we established a high-throughput protein pipeline (HTPP) for the production of a subset of the human secretome. The HTPP was based on a transient expression system in HEK293-EBNA cells at 100 to 500 mL culture scale, combined with an automated affinity purification procedure targeting > 75% purity. This was followed by a semiautomated protein sample logistics to provide biologists with quality-controlled and 96 well formatted protein aliquots amenable to cell-based assays. Over a 4-year period, beginning in 2001, we performed over 7,500 transfections representing over 2,200 registered proteins, including both novel and reference proteins, at an average production of 280 proteins/ month with a peak production of 320 proteins/month. All these proteins have been tested in more than 50 different cell-based assays. This article describes the major process steps and highlights the optimization required to maintain novel protein production while supporting both stock replenishment and scale-up productions.
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Cite this article as:
Battle Thierry, Antonsson Bruno, Feger Georg and Besson Dominique, A High-Throughput Mammalian Protein Expression, Purification, Aliquoting and Storage Pipeline to Assemble a Library of the Human Secretome, Combinatorial Chemistry & High Throughput Screening 2006; 9 (9) . https://dx.doi.org/10.2174/138620706778700143
DOI https://dx.doi.org/10.2174/138620706778700143 |
Print ISSN 1386-2073 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5402 |
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