Affinity Proteomics for Systematic Protein Profiling of Chromosome 21 Gene Products in Human Tissues
2003; Elsevier BV; Volume: 2; Issue: 6 Linguagem: Inglês
10.1074/mcp.m300022-mcp200
ISSN1535-9484
AutoresCharlotta Agaton, Joakim Galli, Ingmarie Höidén Guthenberg, Lars Janzon, Marianne Hansson, Anna Asplund, Eva Brundell, Susanne Lindberg, Irene Ruthberg, Kenneth Wester, Dorothee Wurtz, Christer Höög, Joakim Lundeberg, Stefan Ståhl, Fredrik Pontén, Mathias Uhlén,
Tópico(s)Metabolism and Genetic Disorders
ResumoHere we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues. Here we show that an affinity proteomics strategy using affinity-purified antibodies raised against recombinant human protein fragments can be used for chromosome-wide protein profiling. The approach is based on affinity reagents raised toward bioinformatics-designed protein epitope signature tags corresponding to unique regions of individual gene loci. The genes of human chromosome 21 identified by the genome efforts were investigated, and the success rates for de novo cloning, protein production, and antibody generation were 85, 76, and 56%, respectively. Using human tissue arrays, a systematic profiling of protein expression and subcellular localization was undertaken for the putative gene products. The results suggest that this affinity proteomics strategy can be used to produce a proteome atlas, describing distribution and expression of proteins in normal tissues as well as in common cancers and other forms of diseased tissues. A crucial challenge in the post-genomic era is to utilize the genome information for better understanding of protein expression and function. Most frequently, this has been done using protein separation techniques coupled with mass spectrometry analysis methods (1Patterson S.D. Aebersold R. Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis. 1995; 16: 1791-1814Google Scholar, 2Mann M. Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal. Chem. 1994; 66: 4390-4399Google Scholar, 3Yates 3rd, J.R. Eng J.K. McCormack A.L. Schieltz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 1995; 67: 1426-1436Google Scholar, 4Han D.K. Eng J. Zhou H. Aebersold R. Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry. Nat. Biotechnol. 2001; 19: 946-951Google Scholar). Recently, several methods have been described for "genome-based" proteomics approaches aimed to enumerate and functionally catalogue all the components of the proteome by a gene-by-gene approach. Initially, these studies have involved the analysis of networks of protein interactions, using genetic tools such as two-hybrid systems (5Fields S. Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989; 340: 245-246Google Scholar, 6Chien C.T. Bartel P.L. Sternglanz R. Fields S. The two-hybrid system: A method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Google Scholar, 7Auerbach D. Thaminy S. Hottiger M.O. Stagljar I. The post-genomic era of interactive proteomics: facts and perspectives. Proteomics. 2002; 2: 611-623Google Scholar) and protein complex pull-outs (8Kuster B. Mortensen P. Andersen J.S. Mann M. Mass spectrometry allows direct identification of proteins in large genomes. Proteomics. 2001; 1: 641-650Google Scholar, 9Ho Y. Gruhler A. Heilbut A. Bader G.D. Moore L. Adams S.L. Millar A. Taylor P. Bennett K. Boutilier K. Yang L. Wolting C. Donaldson I. Schandorff S. Shewnarane J. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry. Nature. 2002; 415: 180-183Google Scholar, 10Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature. 2002; 415: 141-147Google Scholar). Furthermore, structural genomics strategies have been developed to allow streamlined unit operations for protein structure determination (11Hammarström M. Hellgren N. van Den Berg S. Berglund H. Härd T. Rapid screening for improved solubility of small human proteins produced as fusion proteinsEscherichia coli. Protein Sci. 2002; 11: 313-321Google Scholar). As a complement to these efforts, a more general tool for genome-based proteomics has been proposed (12Larsson M. Brundell E. Nordfors L. Höög C. Uhlén M. Ståhl S. A general bacterial expression system for functional analysis of cDNA-encoded proteins. Protein Expr. Purif. 1996; 7: 447-457PubMed Google Scholar, 13Larsson M. Gräslund S. Yuan L. Brundell E. Uhlén M. Höög C. Ståhl S. High-throughput protein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 2000; 80: 143-157Google Scholar, 14Gräslund S. Falk R. Brundell E. Höög C. Ståhl S. A high-stringency proteomics concept aimed for generation of antibodies specific for cDNA-encoded proteins. Biotechnol. Appl. Biochem. 2002; 35: 75-82Google Scholar) in which protein-specific affinity reagents are generated, which can be used in a stepwise manner for a wide range of functional and biochemical studies. There are several challenges for such a strategy, here called affinity proteomics. First, the success rates for recombinant expression of human proteins in bacteria are normally relatively low (11Hammarström M. Hellgren N. van Den Berg S. Berglund H. Härd T. Rapid screening for improved solubility of small human proteins produced as fusion proteinsEscherichia coli. Protein Sci. 2002; 11: 313-321Google Scholar). Second, both polyclonal and monoclonal antibodies often give cross-reactivity to other proteins or show high background binding in tissue sections (14Gräslund S. Falk R. Brundell E. Höög C. Ståhl S. A high-stringency proteomics concept aimed for generation of antibodies specific for cDNA-encoded proteins. Biotechnol. Appl. Biochem. 2002; 35: 75-82Google Scholar). Third, high-throughput schemes have been difficult to employ both with regard to automation and cost-efficiency. These problems have seriously hampered whole proteome applications to generate antibodies and have prompted us to try new approaches for protein expression and antibody generation. Here we describe a strategy for affinity proteomics based on the generation of protein epitope signature tags (PrESTs). 1The abbreviation used is: PrEST, protein epitope signature tag. The protein fragments (PrESTs) are designed to contain unique epitopes present in the native protein suitable for triggering the generation of antibodies of high selectivity. A strategy based on the generation of polyclonal antibodies has been followed. The antibodies are affinity purified using the target protein as ligand. The use of polyclonal antibodies, instead of monoclonal antibodies, makes the generation relatively cost-effective and increases the probability of specific recognition of the target protein during a variety of denaturating conditions. Although monoclonal antibodies are attractive for routine use, such as diagnostics, therapeutics, or protein arrays, the use of polyclonal antibodies is well suited for the protein profiling, in which the proteins have been denaturated in different conditions, such as with formalin for the paraffin-imbedded tissues or with SDS for the protein extracts analyzed on Western blots. As a pilot project, we describe an analysis of the putative gene products of human chromosome 21. Although the project was designed to provide a general proof-of-concept for whole proteome analysis, specific information gained from the chromosome 21-encoded proteome may be valuable for the studies of a range of common complex diseases that map to this chromosome, also including disorders such as cancer and Down syndrome that result from deletion or duplication of sequences on this chromosome. All genes of human chromosome 21 (15Hattori M. Fujiyama A. Taylor T.D. Watanabe H. Yada T. Park H.S. Toyoda A. Ishii K. Totoki Y. Choi D.K. Soeda E. Ohki M. Takagi T. Sakaki Y. Taudien S. The DNA sequence of human chromosome 21. Nature. 2000; 405: 311-319Google Scholar) with open reading frames larger than 100 amino acid residues were subjected to PrEST design and production. A set of PrESTs representing proteins with both known and unknown function were selected for further analysis, and antibodies were generated and affinity-purified using the PrESTs as ligands. The results suggest that this affinity proteomics strategy can be used for systematic generation of protein profiles and subcellular localization data on a genome-wide level. RT-PCR was performed using Superscript One Step RT-PCR with Platinum Taq (Life Technologies, Rockville, MD) using a human Total RNA Panel IV (CLONTECH, Palo Alto, CA) as template. Flanking restriction sites NotI and AscI, respectively, were introduced into the fragments through the specific primers to allow "in frame" cloning into the expression vector pAff8c (13Larsson M. Gräslund S. Yuan L. Brundell E. Uhlén M. Höög C. Ståhl S. High-throughput protein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 2000; 80: 143-157Google Scholar). The downstream primer was biotinylated to allow solid-phase cloning as previously described (13Larsson M. Gräslund S. Yuan L. Brundell E. Uhlén M. Höög C. Ståhl S. High-throughput protein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 2000; 80: 143-157Google Scholar). The resulting biotinylated PCR products were immobilized onto Dynabeads M280-streptavidin (Dynal Biotech, Oslo, Norway). The fragments were released from the solid support by NotI-AscI digestion, ligated into pAff8c, and transformed into Escherichia coli BL21(DE3) cells. The sequences of the clones were verified by dye-terminator cycle sequencing of purified plasmid DNA. BL21(DE3) cells, harboring the different expression constructs, were inoculated into culture medium as described before (13Larsson M. Gräslund S. Yuan L. Brundell E. Uhlén M. Höög C. Ståhl S. High-throughput protein expression of cDNA products as a tool in functional genomics. J. Biotechnol. 2000; 80: 143-157Google Scholar) and induced at A600 nm ≈ 1.0 with isopropyl-β-d-thiogalactopyranoside (Sigma Aldrich, St. Louis, MO) at a final concentration of 1 mm, and the incubation was continued overnight at 25 °C. The cells were harvested by centrifugation, and the pellet was resuspended in 5 ml lysis buffer (7 m guanidinium-HCl, 47 mm Na2HPO4, 2.65 mm NaH2PO4, 10 mm Tris-HCl, 100 mm NaCl, pH 8.0, 20 mm β-mercaptoethanol) and incubated for 2 h at 37 °C. The solutions were sheared with a bore 0.8-mm needle (Becton Dickinson, Franklin Lakes, NJ) and a 10-ml syringe (Becton Dickinson) to reduce the viscosity. After centrifugation, the supernatants, containing the denatured and solubilized gene products, were filtered (0.45 μm, Sartorius AG, Goettingen, Germany) prior to the immobilized metal ion affinity chromatography procedure. The immobilized metal ion affinity chromatography purification of the His6-tagged fusion proteins was performed on TALON metal (Co2+) affinity resins and gravity columns (CLONTECH) as recommended by the manufacturer. New Zealand rabbits were immunized with the purified PrEST fragments by AgriSera AB (Vännäs, Sweden) in accordance with the national guidelines (Swedish permit no.A125/00). The rabbits were immunized subcutaneously with 200 μg of antigen in Freund's complete adjuvant as the primary immunization and boosted three times in 3-week intervals with 100 μg of antigen in Freund's incomplete adjuvant. Enrichment of antibodies reactive to the fusion proteins was performed as described previously (16Starborg M. Höög C. The murine replication protein P1 is differentially expressed during spermatogenesis. Eur. J. Cell Biol. 1995; 68: 206-210Google Scholar). Western blots with proteins extracted from 14 different human tissues were prepared by separation on Novex Nupage gels (4–12%, 4-morpholineethanesulfonic acid buffer system, Novex, Invitrogen, San Diego, CA) followed by transfer to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Bedford, MA) in Nupage transfer buffer (according to the manufacturer's protocol). The human tissues were skin (SK), breast (BR), omental fat (OF), tonsil (TO), placenta (PL), lung (LU), kidney, (KI), liver (LI), gallbladder (GB), colon (CO), ileum (IL), duodenum (DU), ventricle (VE), and esophagus (ES). The membranes were incubated with the affinity-enriched affinity reagents (1:75) and washed and probed with horseradish peroxidase-conjugated anti-rabbit IgG (Sigma). SuperSignal West Dura (Pierce, Rockford, IL) was used as detection system, and digital images of the chemiluminescense were monitored using a ChemiImager (Alpha Innotech, San Leandro, CA). A wide spectrum of normal and neoplastic tissues were chosen from 52 donor blocks, all selected from the archives at the Department of Pathology, University Hospital, Uppsala. Selection of normal tissue was based on morphological criteria, and samples were taken from morphologically normal areas surrounding diseased tissue, e.g. liver was chosen from a block containing normal liver outside a liver metastasis, heart from morphologically normal myocardial tissue from patient with cardiomyopathy, etc. In all, 43 such normal tissues were chosen as representative of normal human organs and tissues. Nine samples from different tumors were also included. Using a Tissue arrayer (Beecher Instruments, Silver Spring, MD), three 1.0-mm punches were acquired from defined areas of each selected tissue. The final tissue array block contained 156 (12 × 13) tissue cylinders. Paraffin sections of 4-μm thickness were placed onto Superfrost/plus® slides (Roche Applied Science, Basel, Switzerland), deparaffinized in xylene, and rehydrated in graded alcohols. For antigen retrieval, slides were immersed in 0.01 m citrate buffer, pH 6.0, and boiled for 7 min in a Decloacing chamber® (Biocare Medical, Walnut Creek, CA). Alternatively, slides were treated with 0.05% protease type VIII (Sigma) for 10 min at room temperature. Endogenous peroxidase was blocked in 0.3% H2O2 in PBS for 20 min. To reduce nonspecific binding of the primary antisera, sections were pre-incubated in 0.5% BSA-c® (Aurion, Wageningen, The Netherlands) in PBS. The purified affinity reagents, diluted 1:400, were used as primary antibodies, and the sections were incubated overnight at 4 °C. Incubation with anti-rabbit peroxidase-conjugated Envision® (DAKO, Copenhagen, Denmark) was done at room temperature for 30 min. Diaminobenzidine (Sigma) was used as chromogen, and Harris hematoxylin (Sigma) was used for counterstaining. Parallel slides were immunostained both manually and in an autostainer (Ventana Medical Systems, Tucson, Arizona). As a starting point, we performed bioinformatics analysis of all the putative genes of human chromosome 21 found by Hattori et al. (15Hattori M. Fujiyama A. Taylor T.D. Watanabe H. Yada T. Park H.S. Toyoda A. Ishii K. Totoki Y. Choi D.K. Soeda E. Ohki M. Takagi T. Sakaki Y. Taudien S. The DNA sequence of human chromosome 21. Nature. 2000; 405: 311-319Google Scholar). Out of the 225 putative genes mapped to the chromosome, 168 of them were found to contain an open reading frame longer than 100 amino acids (300 base pairs). The results are summarized in Fig. 1. Sequence analysis algorithms were used to select suitable PrEST coding regions for all of these genes. Transmembrane spanning regions were identified using transmembrane hidden Markow model algorithms (17Sonnhammer E.L. von Heijne G. Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1998; 6: 175-182PubMed Google Scholar, 18Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 2001; 305: 567-580Google Scholar), and these regions were avoided in the selection of PrESTs. To minimize cross-reactivity of the affinity reagents, regions and domains with homology to other human proteins were omitted using homology search programs, such as BLAST (19Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar). The protein fragments, with a size of 100–150 residues, with the lowest homology to other proteins in the human proteome were thus selected. A total of 168 PCR primer pairs were designed to flank the selected (PrEST) regions. To clone the PrESTs, we used a pool of RNA from various human tissues in an RT-PCR approach. Use of a single RNA pool consisting of material from human brain, spinal cord, placenta, and liver proved to be a valuable approach allowing us to amplify 120 out of the 168 genes (71%) in a first attempt. By designing new primers for those genes that failed, a second round yielded PCR products from 22 additional genes. The overall success rate for the two rounds of RT-PCR was thus 142 out of 168 genes (85%). The 142 amplified gene fragments were cloned into an expression vector in frame with a dual affinity tag consisting of a hexahistidyl tag, allowing purification on nickel columns, in frame with an immunopotentiating albumin binding domain (20Ståhl, S., Nilsson J., Hober, S., Uhlén, M., and Nygren PÅ. 1999. Affinity fusions, gene expression, in Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation (Flickinger, M. C., and Drew, S. W., ed.) pp. 8–22, John Wiley & Sons, New YorkGoogle Scholar). All of the amplification products were successfully cloned and sequence verified. The E. coli clones expressed in liquid cultures were lysed in the presence of guanidinium chloride, and the cleared lysates were passed over nickel-containing matrices. In a majority of cases, more than 0.5 mg of proteins of the right size was obtained from 50-ml cultures. A summary of the results of all the putative chromosome 21 genes is shown in Fig. 1. The overall success rate for the protein production using a single expression event was 108 out of 142 (76%). Two rabbits were immunized with each of the purified PrESTs and the polyclonal antisera were individually affinity purified using the specific PrESTs as ligands to obtain PrEST-specific antibodies. As a first step in the protein expression analysis, and to validate functionality of the antibodies, screening for expression of the various gene products was performed using protein extracts from a panel of human tissues in a Western blotting format. In Fig. 2, the results before and after affinity purification are shown for a protein with known function (SOD1) and a protein with unknown function (KIAA0539). In both cases, the affinity purification yields a protein pattern with bands of the expected sizes (16 kDa and 252 kDa, respectively). It is noteworthy that several smaller bands displaying the same tissue distribution were obtained using the KIAA0539 affinity reagent. Plausible explanations for this could be the existence of splice variants, proteolysis, protein modifications, such as glycosylation, or simply cross-reactivity of the affinity reagent. The difference in size makes this protein interesting for further studies to also analyze the possibility of mRNA splice variants or protein modifications. In Fig. 3, examples from eight antibodies with known (CCT8, SOD1, CBR1, and ATP5O) and unknown (KIAA0539, SAMSN-1, WDR9, and ZNF294) function are shown. For the four known gene products (Fig. 3, A–D), a band of the expected size was obtained in all cases. Furthermore, the expression profiles of the known genes were in accordance with previously published data (21Golden T.R. Pedersen P.L. The oligomycin sensitivity conferring protein of rat liver mitochondrial ATP synthase: Arginine 94 is important for the binding of OSCP to F1. Biochemistry. 1998; 37: 13871-13881Google Scholar, 22Forrest G.L. Gonzalez B. Carbonyl reductase. Chem. Biol. Interact. 2000; 129: 21-40Crossref PubMed Scopus (209) Google Scholar, 23Brown C.R. Doxsey S.J. Hong-Brown L.Q. Martin R.L. Welch W.J. Molecular chaperones and the centrosome. A role for TCP-1 in microtubule nucleation. J. Biol. Chem. 1996; 271: 824-832Google Scholar). For the four gene products of unknown function, bands with expected sizes were also detected, as deduced from the amino acid sequences (Fig. 3, E–H). A summary of the results of the tissue Western analysis is shown in Fig. 1. The overall success rate of affinity-purified antibodies as determined by a specific band in the tissue Western analysis was 54/96 (56%).Fig. 3Tissue distribution of the gene products as determined by Western blot using protein extracts from human tissues. The order of the tissues is the same as in Fig. 2. CCT8, SOD1, CBR1, ATP5O, KIAA0539, SAMSN-1, WDR9, and ZNF294 were detected using the PrEST specific affinity reagents. The molecular mass of each gene product deduced from the amino acid sequence is given.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To allow for high-throughput protein profiling, tissue arrays consisting of a variety of human tissues were constructed for analysis of cellular localization of the gene products. The arrays were produced by transferring cylinders of tissues from a multitude of donor blocks consisting of archival paraffin-embedded tissues to a single recipient paraffin block (24Rimm D.L. Camp R.L. Charette L.A. Costa J. Olsen D.A. Reiss M. Tissue microarray: A new technology for amplification of tissue resources. Cancer J. 2001; 7: 24-31Google Scholar, 25Kononen J. Bubendorf L. Kallioniemi A. Barlund M. Schraml P. Leighton S. Torhorst J. Mihatsch M.J. Sauter G. Kallioniemi O.P. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med. 1998; 4: 844-847Google Scholar). The recipient block was subsequently used to produce a large number of slides containing 52 different tissues as triplicate spots with a diameter of 1.0 mm (Fig. 4). The tissue array permitted simultaneous analysis of both cellular and subcellular localization of expressed proteins in more than 50 different tissues. Fig. 4 shows an example of the tissue array analysis for the unknown human protein KIAA0539. The analysis showed that the protein was expressed in glandular cells of the colon and in smooth muscle cells beneath the colonic mucosa. A more detailed analysis of crypts revealed that the protein was localized in the Goblet cell vacuoles. In Fig. 5, illustrative examples of the tissue array results of affinity reagents to the eight selected gene products are shown. The protein profiles of the four gene products having known function are well in accordance with the intracellular and tissue distribution suggested by earlier studies. For example, the detoxification enzyme, superoxide dismutase (SOD1), was highly expressed in the liver, whereas ATP5O, a subunit of the ATP synthetase in the mitochondrial F1 complex, was expressed in virtually all cells and visualized with an idiosyncratic microgranular staining pattern. The affinity reagents recognizing proteins with unknown function gave distinct staining patterns yielding specific expression and localization profiles. ZNF294, for example, shows a predominantly cytoplasmic staining with variable distribution in different tissues. A strong immunostaining was found in testis, where seminiferous tubules including germinative epithelia and Sertoli as well as Leydig cells reacted with the ZNF294 affinity reagents. A crescent-shaped immunoreactivity was found in spermatids, although mature sperms appeared negative. The WDR9 affinity reagent was negative in a majority of tissues, but a focal and distinct granular immunoreactivity was found in hepatocytes and zona reticulosa cells in the adrenal gland. The distribution and pattern of immunoreactivity resemble that of lipofuscin and suggest that WDR9 has some relation to the lysosomal deposition of lipofuscin. KIAA0539 was expressed by mucus-producing tissues, such as colon and the small intestine and the uterine cervical mucosa. In addition, smooth muscle cells of the prostatic gland showed high KIAA0539 expression. In Fig. 6, the expression patterns for 12 different gene products are shown for two brain tissues (cerebrum and cerebellum). Distinct protein expression profiles can be observed, such as the dendritic/axonal positivity of MCM3, the expression of ZNF294 and C21orf33 in Bergman astrocytes, and the mosaic like pattern of GABPA in cerebrum. Here we have analyzed the putative genes predicted from the genome sequence of chromosome 21. Chromosome 21 was chosen to establish the applicability of a chromosome-wide study. The original 225 genes described by Hattori et al. (15Hattori M. Fujiyama A. Taylor T.D. Watanabe H. Yada T. Park H.S. Toyoda A. Ishii K. Totoki Y. Choi D.K. Soeda E. Ohki M. Takagi T. Sakaki Y. Taudien S. The DNA sequence of human chromosome 21. Nature. 2000; 405: 311-319Google Scholar) were included in this pilot study, although recent predictions have indicated a slightly larger gene content of 238 genes (26Gitton Y. Dahmane N. Baik S. Ruiz i Altaba A. Neidhardt L. Scholze M. Herrmann B.G. Kahlem P. Benkahla A. Schrinner S. Yildirimman R. Herwig R. Lehrach H. Yaspo M.L. A gene expression map of human chromosome 21 orthologues in the mouse. Nature. 2002; 420: 586-590Google Scholar). We chose to analyze proteins with an open reading frame larger than 100 amino acids, but obviously smaller proteins could be included in an extended study. Using a combination of bioinformatics, recombinant protein expression, and cost-effective antibody production, we have generated a large set of affinity ligands in the form of monospecific polyclonal antibodies. We show that these affinity-purified antibodies are useful tools to explore protein expression profiles using human tissue arrays. Together, these methodologies allow for a systematic approach to generate and use affinity reagents without dependence on clone repositories or cumbersome expression screening procedures. The affinity proteomics strategy is based on the generation of PrESTs, which are amenable both to recombinant protein expression and the generation of specific antibody reagents. The availability of the whole genome sequence allows the most unique (nonhomologous) region within a specific protein to be selected for protein expression. Comparative algorithms are used to ensure that highly homologous protein domains in the human proteome are not being used for protein expression and thus not included as immunogens during immunization. Second, transmembrane regions are omitted, because they are difficult to express and purify (27Miroux B. Walker J.E. Over-production of proteins in Escherichia coli: Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 1996; 260: 289-298Google Scholar) and are not likely to be suited for immunolocalization studies due to less accessibility for the affinity reagents. Third, protein fragments of 100–150 amino acid residues are chosen to facilitate cloning and protein expression and to possibly provide conformational epitopes not obtained using shorter peptides. In most cases, the PrESTs will not have a fully native fold. However, the fact that the subsequent protein profiling is performed on denaturated proteins makes it attractive to generate affinity reagents toward partly denaturated proteins. It is in this context an advantage to use polyclonal affinity reagents with a multiple of binding epitopes to increase the probability that some epitopes are present during the conditions for the different protein profiling procedures. An important finding is that a single pool of RNA extracted from four human tissues can be used for efficient RT-PCR cloning. Using this strategy, rapid cloning was performed without dependence on availability of clones through various clone repositories. This is of particular importance for whole proteome efforts, because many of the putative genes are solely predicted from the genome sequence. An alternative would be to use chromosomal DNA as a source of genes. However, the size of an average human exon is only 48 amino acids (28Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. Initial sequencing and analysis of the human genome. Nature. 2001; 409: 860-921Google Scholar), and the production of PrESTs would require a cumbersome assembly of several exons using "PCR splicing" techniques (29Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene. 1989; 77: 61-68Google Scholar, 30Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989; 77: 51-59Google Scholar). The genes encoding the PrEST regions can also be
Referência(s)