Proteome-scale purification of human proteins from bacteria
Open Access
- 5 March 2002
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 99 (5) , 2654-2659
- https://doi.org/10.1073/pnas.042684199
Abstract
The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequence-verified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character.Keywords
This publication has 27 references indexed in Scilit:
- FLEXGene repository: from sequenced genomes to gene repositories for high-throughput functional biology and proteomicsMolecular and Biochemical Parasitology, 2001
- Global Efforts in Structural GenomicsScience, 2001
- Global Analysis of Protein Activities Using Proteome ChipsScience, 2001
- Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fusedProtein Science, 1999
- Affinity Fusion Strategies for Detection, Purification, and Immobilization of Recombinant ProteinsProtein Expression and Purification, 1997
- High-Level Expression of Soluble Protein inEscherichia coliUsing a His6-Tag and Maltose-Binding-Protein Double-Affinity Fusion SystemProtein Expression and Purification, 1997
- New functional activities for the p21 family of CDK inhibitors.Genes & Development, 1997
- Upstream Strategies to Minimize Proteolytic Degradation upon Recombinant Production inEscherichia coliProtein Expression and Purification, 1996
- Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chainsProtein Science, 1995
- Recent developments in heterologous protein production in Escherichia coliTrends in Biotechnology, 1994