High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins.

Modern proteomics approaches include techniques to examine the expression, localization, modifications, and complex formation of proteins in cells. In order to address issues of protein function in vitro using classical biochemical and biophysical approaches, high-throughput methods of cloning the appropriate reading frames, and expressing and purifying proteins efficiently are an important goal of modern proteomics approaches. This process becomes more difficult as functional proteomics efforts focus on the proteins from higher organisms, since issues of correctly identifying intron-exon boundaries and efficiently expressing and solubilizing the (often) multi-domain proteins from higher eukaryotes are challenging. Recently, 12,000 open-reading-frame (ORF) sequences from Caenorhabditis elegans have become available for functional proteomics studies [Nat. Gen. 34 (2003) 35]. We have implemented a high-throughput screening procedure to express, purify, and analyze by mass spectrometry hexa-histidine-tagged C. elegans ORFs in Escherichia coli using metal affinity ZipTips. We find that over 65% of the expressed proteins are of the correct mass as analyzed by matrix-assisted laser desorption MS. Many of the remaining proteins indicated to be "incorrect" can be explained by high-throughput cloning or genome database annotation errors. This provides a general understanding of the expected error rates in such high-throughput cloning projects. The ZipTip purified proteins can be further analyzed under both native and denaturing conditions for functional proteomics efforts.

[1]  M. Vidal,et al.  Combined Functional Genomic Maps of the C. elegans DNA Damage Response , 2002, Science.

[2]  Adam Godzik,et al.  Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Chance,et al.  Determination of macromolecular folding and structure by synchrotron x-ray radiolysis techniques. , 2001, Analytical biochemistry.

[4]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[5]  S. Almo,et al.  Mapping the G-actin binding surface of cofilin using synchrotron protein footprinting. , 2002, Biochemistry.

[6]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[7]  H. Bedouelle,et al.  Production in Escherichia coli and one-step purification of bifunctional hybrid proteins which bind maltose. Export of the Klenow polymerase into the periplasmic space. , 1988, European journal of biochemistry.

[8]  M. Mann,et al.  Analysis of proteins and proteomes by mass spectrometry. , 2001, Annual review of biochemistry.

[9]  B. Slatko,et al.  An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. , 1988, Gene.

[10]  Eric Gouaux,et al.  A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase , 1999, Protein science : a publication of the Protein Society.

[11]  M. Vidal,et al.  Structural genomics: A pipeline for providing structures for the biologist , 2002, Protein science : a publication of the Protein Society.

[12]  F. Baneyx.  Recombinant protein expression in Escherichia coli. , 1999, Current opinion in biotechnology.

[13]  S. Fields,et al.  Array-based methods for identifying protein-protein and protein-nucleic acid interactions. , 2002, Methods in enzymology.

[14]  R. Hettich,et al.  Protein surface mapping by chemical oxidation: structural analysis by mass spectrometry. , 2003, Analytical biochemistry.

[15]  J. Hudson,et al.  The complete set of predicted genes from Saccharomyces cerevisiae in a readily usable form. , 1997, Genome research.

[16]  M. Chance,et al.  Solution structure and interdomain interactions of the Saccharomyces cerevisiae "TATA binding protein" (TBP) probed by radiolytic protein footprinting. , 2003, Biochemistry.

[17]  M. Chance,et al.  Unfolding of apomyoglobin examined by synchrotron footprinting. , 2001, Biochemical and biophysical research communications.

[18]  P. Janmey,et al.  Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Vidal,et al.  GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. , 2000, Methods in enzymology.

[20]  D. Smith,et al.  Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. , 1988, Gene.

[21]  Scott A Lesley,et al.  Gene expression response to misfolded protein as a screen for soluble recombinant protein. , 2002, Protein engineering.

[22]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[23]  J. Hudson,et al.  C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression , 2003, Nature Genetics.

[24]  M Chance,et al.  Synchrotron Protein Footprinting: A Technique to Investigate Protein-Protein Interactions , 2001, Journal of biomolecular structure & dynamics.

[25]  Peter Roepstorff,et al.  Electrospray ionization and matrix assisted laser desorption/ionization mass spectrometry: Powerful analytical tools in recombinant protein chemistry , 1996, Nature Biotechnology.

[26]  R. Jaenicke,et al.  A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. , 1991, Biochemistry.

[27]  B. Chait,et al.  Mapping protein-protein interactions by affinity-directed mass spectrometry. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  M. Chance,et al.  Hydroxyl radical probe of protein surfaces using synchrotron X-ray radiolysis and mass spectrometry , 2002, International journal of radiation biology.