Generation of High Density Protein Microarrays by Cell-free in Situ Expression of Unpurified PCR Products

Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 μg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (∼22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones. Due to the success of DNA microarrays and the growing numbers of available protein expression clones, protein microarrays have become more and more popular for the high throughput screening of protein interactions. However, the widespread applicability of protein microarrays is currently hampered by the large effort associated with their production. Apart from the requirement for a protein expression library, expression and purification of the proteins themselves and the lacking stability of many proteins remain the bottleneck. Here we present an approach that allows the generation of high density protein microarrays from unbound DNA template molecules on the chip. It is based on the multiple spotting technique and comprises the deposition of a DNA template in a first spotting step and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting step. Using wild-type green fluorescent protein as a model protein, we demonstrated the time and template dependence of this coupled transcription and translation and showed that enough protein was produced to yield signals that were comparable to 300 μg/ml spotted protein. Plasmids as well as unpurified PCR products can be used as templates, and as little as 35 fg of PCR product (∼22,500 molecules) were sufficient for the detectable expression of full-length wild-type green fluorescent protein in subnanoliter volumes. We showed that both aminopropyltrimethoxysilane and nickel chelate surfaces can be used for capture of the newly synthesized proteins. Surprisingly we observed that nickel chelate-coated slides were binding the newly synthesized proteins in an unspecific manner. Finally we adapted the system to the high throughput expression of libraries by designing a single primer pair for the introduction of the required T7 promoter and demonstrated the in situ expression using 384 randomly chosen clones. The understanding of complex cellular networks necessitates tools that are amenable to the analysis of different parameters in a highly parallel manner (1Hoheisel J.D. Microarray technology: beyond transcript profiling and genotype analysis.Nat. Rev. Genet. 2006; 7: 200-210Crossref PubMed Scopus (425) Google Scholar). Although in the last years DNA microarrays were the technology of choice to monitor the abundance of several thousands of mRNA transcripts at a time, such studies provide us with little information on the proteins that are encoded by these transcripts (2Griffin T.J. Gygi S.P. Ideker T. Rist B. Eng J. Hood L. Aebersold R. Complementary profiling of gene expression at the transcriptome and proteome levels in Saccharomyces cerevisiae.Mol. Cell. Proteomics. 2002; 1: 323-333Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 3Hack C.J. Integrated transcriptome and proteome data: the challenges ahead.Brief. Funct. Genomics Proteomics. 2004; 3: 212-219Crossref PubMed Scopus (124) Google Scholar). However, because proteins rather than DNA carry out cellular functions, there is large interest to analyze proteins and their entirety, the proteome, in a manner comparable to DNA microarrays. One technology that is envisaged to meet the demands of high throughput protein interaction and modification screening is protein microarray technology (4Bertone P. Snyder M. Advances in functional protein microarray technology.FEBS. J. 2005; 272: 5400-5411Crossref PubMed Scopus (144) Google Scholar, 5Merkel J.S. Michaud G.A. Salcius M. Schweitzer B. Predki P.F. 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Functional protein microarrays for parallel characterisation of p53 mutants.Proteomics. 2004; 4: 1950-1958Crossref PubMed Scopus (73) Google Scholar, 10Zhu H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Global analysis of protein activities using proteome chips.Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1942) Google Scholar, 11Schweitzer B. Predki P. Snyder M. Microarrays to characterize protein interactions on a whole-proteome scale.Proteomics. 2003; 3: 2190-2199Crossref PubMed Scopus (138) Google Scholar), the identification of substrates for protein kinases (12Feilner T. Hultschig C. Lee J. Meyer S. Immink R.G. Koenig A. Possling A. Seitz H. Beveridge A. Scheel D. Cahill D.J. Lehrach H. Kreutzberger J. Kersten B. High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.Mol. Cell. 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Profiling humoral autoimmune repertoire of dilated cardiomyopathy (DCM) patients and development of a disease-associated protein chip.Proteomics. 2006; 6: 605-613Crossref PubMed Scopus (73) Google Scholar, 16Steller S. Angenendt P. Cahill D.J. Heuberger S. Lehrach H. Kreutzberger J. Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.Proteomics. 2005; 5: 2048-2055Crossref PubMed Scopus (53) Google Scholar, 17Feng Y. Ke X. Ma R. Chen Y. Hu G. Liu F. Parallel detection of autoantibodies with microarrays in rheumatoid diseases.Clin. Chem. 2004; 50: 416-422Crossref PubMed Scopus (66) Google Scholar, 18Robinson W.H. DiGennaro C. Hueber W. Haab B.B. Kamachi M. Dean E.J. Fournel S. Fong D. Genovese M.C. de Vegvar H.E. Skriner K. Hirschberg D.L. Morris R.I. Muller S. Pruijn G.J. van Venrooij W.J. Smolen J.S. Brown P.O. Steinman L. Utz P.J. Autoantigen microarrays for multiplex characterization of autoantibody responses.Nat. Med. 2002; 8: 295-301Crossref PubMed Scopus (643) Google Scholar). All of them share the basic principle of production. It involves the generation of expression clones and the subsequent expression and purification of proteins off the chip followed by spotting them onto the microarray surface. Currently this process represents a major bottleneck in the production of protein microarrays because both the generation of the expression clones and the purification of the proteins are time- and cost-intensive even in low throughput applications (19Glokler J. Angenendt P. Protein and antibody microarray technology.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003; 797: 229-240Crossref PubMed Scopus (139) Google Scholar, 20Labaer J. Ramachandran N. Protein microarrays as tools for functional proteomics.Curr. Opin. Chem. Biol. 2005; 9: 14-19Crossref PubMed Scopus (240) Google Scholar). To circumvent this bottleneck, different approaches have been developed for the generation of protein microarrays. Madoz-Gurpide et al. prepared cell lysate from adenocarcinoma cell lines and fractionated the protein extract first by anion exchange and then by reverse phase liquid chromatography (21Madoz-Gurpide J. Wang H. Misek D.E. Brichory F. Hanash S.M. Protein based microarrays: a tool for probing the proteome of cancer cells and tissues.Proteomics. 2001; 1: 1279-1287Crossref PubMed Scopus (134) Google Scholar). The obtained fractions were then spotted and probed with antibodies against different proteins. Although this approach is truly advantageous with regard to proper post-translational modifications of the proteins, only mixtures of protein are displayed in which less abundant proteins may not be detectable. He and Taussig (22He M. Taussig M.J. Single step generation of protein arrays from DNA by cell-free expression and in situ immobilisation (PISA method).Nucleic Acids Res. 2001; 29: 73Crossref PubMed Scopus (192) Google Scholar, 23He M. Taussig M.J. DiscernArray technology: a cell-free method for the generation of protein arrays from PCR DNA.J. Immunol. Methods. 2003; 274: 265-270Crossref PubMed Scopus (44) Google Scholar) introduced the idea of creating protein arrays by cell-free transcription and translation of PCR products and the subsequent purification on nickel chelate-coated surfaces. However, their approach, termed “protein in situ array” is by the large of used for the expression and by the capture process that is not on microarrays on nickel chelate-coated or of cell-free transcription and translation as an step the expression on the chip was by Angenendt et al. P. L. W. J. Lehrach H. Cahill D.J. 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It the of the of as on the in with an capture The chip is then with a cell-free expression which expression of the protein and capture of the protein by the antibody the The process is to the of and allows the production of proteins just to the microarray associated with the of protein microarrays. is by several that the gene of interest as a necessitates of on the of the which a of the at to from the or a of transcription by the only low density protein microarrays with to can be produced because a density proteins to to capture antibodies of To these an technology meet several be from a of different templates, PCR be and The generation of protein microarrays be a time just to their to of binding the production of high density protein microarrays of several proteins on a single Here we present an approach that these It is based on the multiple spotting technique P. J. Lehrach H. Cahill D.J. protein microarrays: multiplex on a single Chem. 2003; PubMed Scopus Google Scholar) and comprises the spotting of a DNA template in a first spotting and the transfer of a cell-free transcription and translation mixture on top of the same spot in a second spotting Using this high density protein microarrays with to can be produced from a of different in an and manner. The was obtained from The used for expression is an of the and is based on the a gene wild-type green fluorescent protein to a PCR were synthesized by PCR was from was from and were from of the in were prepared from to The as well as a only were spotted on aminopropyltrimethoxysilane slides as W. A. A. F. J.D. microarrays: an of production 2003; 3: PubMed Scopus Google Scholar) using a spotting system with a with a The that was spot was spotting of the DNA the in transcription and translation mixture was prepared template DNA as by the and spotted onto the same as the template the cell-free transcription and translation mixture was spotted on to which only been the slides were in and at in a for different the slides were in for on a at The slides were with and for in μg/ml antibody in the slides with the slides were for in and by was with a Life Analysis was using All used for analysis were from which the been PCR the of were by PCR of the PCR was in in PCR with the reverse The of the was and of were in a at the at for of for for and for and a step at for PCR products were on and using a PCR purification DNA was by using a of the PCR were prepared from to using PCR mixture were by with for from to μg/ml were prepared by obtained with and and detection were as in the with the chelate-coated slides were used as a the used for the was was at for the slides were in in and detection was by of the slides in μg/ml in for was for in and the slides were with lacking the was using The was and the was by PCR of the and was using the same and as The PCR products were using a PCR purification and PCR products were to in and DNA was as in the using a mixture of μg/ml and μg/ml in 384 were randomly chosen out of a K. C. Lehrach H. G. for protein expression 2000; PubMed Scopus Google Scholar). were in yeast μg/ml μg/ml and and at of in PCR were with of bacterial PCR was with the reverse The of the was of were in a at the at for of for for for and for to and DNA were to the PCR and detection were as of the of Protein expression in was with clones in a of in PCR The transcription and translation mixture was prepared as by the and unpurified PCR products in were used as of the mixture was at in a followed by spotting of the mixture on nickel chelate-coated The slides were at and detection was as of the of The in situ protein expression on the chip is based on the of on spot of the microarrays by a first DNA are spotted on an top of the DNA templates, a cell-free transcription and translation mixture is spotted in a second spotting the is in a which the to and the expression to the is and the proteins are by of DNA mRNA and subsequent translation protein are a coupled by T7 and a complex of the an the of products that is produced is on the of DNA template and To this a of was applied to different microarrays. cell-free transcription and translation mixture was spotted on and the slides were for different of time, of the expression the was the of and time was and the of the template was The an of with of template molecules for the time an of signals with of to was there was a of such as The of was for signals and was to be in the of To the of that are produced and to that unpurified PCR products can be used as DNA templates, the gene for was by and a was a first spotting the PCR products were spotted on nickel chelate-coated the cell-free expression mixture was a of was spotted on the same of slides for was using an which the of both the and produced of the of the only the of full-length was To be to the signals from the expression of and the was that the binding of both the newly synthesized and the were of spotted PCR template protein was prepared by of the of the of the expression and them the The the of unpurified PCR products for expression on a chip with a of the signals DNA full-length in the of 300 of spotted PCR template protein The signals obtained in were and using the of of a in the expression on the chip because provide an that by DNA and cell-free transcription and translation mixture on the and on the the newly synthesized protein to as well as nickel chelate were and both well and with nickel chelate signals not To this was to the capture of the or to unspecific a lacking the was were by PCR and spotted on nickel chelate-coated was by an antibody and an of both displayed comparable for the only the with the signals with the 384 clones from a expression were randomly chosen to the principle of the expression of proteins be to the high throughput expression of proteins from an expression The clones were in the expression that a promoter and for protein the were by a T7 and an promoter for transcription in and To the available for cell-free were from the by PCR using a single primer pair a T7 promoter with the binding and of the T7 to a of the promoter as well as the T7 promoter and the in a for spotting purification of the PCR The DNA were spotted in and detection was their and an antibody of clones high and clones expression was out in to the expression of clones was to the microarray the mixture was spotted on nickel chelate and an antibody was applied for All of the clones high expression on a chip were in the and of the clones expression on the chip displayed a detectable expression in the The generation of high density protein microarrays is a and for this are not the spotting process the production of the proteins, which the generation of expression clones and the subsequent expression and purification of the proteins. Currently the only to this is the which was introduced by Ramachandran et al. N. B. S. B. J. protein 2004; PubMed Scopus Google Scholar). Although this approach a more than the expression and purification of the proteins off the is by several the generation of an expression library, which the as of a T7 these have to be and at the density of the protein microarrays is to because at proteins to To these and to the generation of high density protein microarrays with we developed an for an in situ expression of proteins. a of the expression of a on the chip was To the dependence on time and of different of the were applied to the and the microarrays were for different of a slides with were used because to proteins in microarray applications W. A. A. F. J.D. microarrays: an of production 2003; 3: PubMed Scopus Google Scholar, P. J. Murphy D. Lehrach H. Cahill D.J. antibody microarrays: a of microarray 2002; PubMed Scopus Google Scholar, P. J. J. Lehrach H. Cahill D.J. generation of protein microarray for protein and antibody microarray Chromatogr. A. 2003; PubMed Scopus Google Scholar). an of was observed with of as well as of to the signals may be by by of the cell-free transcription and translation The in which of DNA template was in low to of to To the of using unpurified PCR products and to the yield of the gene was by and different of PCR products were applied to nickel chelate-coated a of was spotted as a an antibody was used that the of the only full-length protein. To be to the of protein that is and to the signals of the expression with the obtained from the we that the binding of the newly synthesized and the are both the were and a of protein spotted PCR template was The that as little as PCR product can be used for expression and that to 300 μg/ml protein or can be produced on the to be that the present in to the and the synthesized not have binding can be that the more to the than the newly synthesized the to with an of proteins from the cell-free transcription and translation that even more have been produced that of protein can be of the newly synthesized onto the is a step in the in situ and nickel were and both demonstrated their Although in the of the the of binding is to be unspecific we to the binding of to the nickel chelate is to an binding the and the nickel chelate microarray as applied in protein purification H. A. chelate for proteins and Chromatogr. PubMed Scopus Google Scholar, nickel chelate for PubMed Scopus Google Scholar). The nickel chelate been to proteins in a and manner in a microarray H. Bilgin M. Bangham R. Hall D. Casamayor A. Bertone P. Lan N. Jansen R. Bidlingmaier S. Houfek T. Mitchell T. Miller P. Dean R.A. Gerstein M. Snyder M. Global analysis of protein activities using proteome chips.Science. 2001; 293: 2101-2105Crossref PubMed Scopus (1942) Google Scholar, J. M. C. of complex detection of low abundant using Res. 2006; 5: PubMed Scopus Google Scholar, C. C. J. K. Microarrays based on detection of in complex 2005; Scholar). However, to not been demonstrated that the binding is truly on the To a was that was lacking the expression on the chip was and the expression products were by an antibody and antibody Although the signals from the antibody were the from the antibody was only detectable for the the that unspecific binding of the protein is the major of binding rather than a and the may be to interactions because high are for chromatography to binding of proteins. To the expression on a chip can be applied to proteins than 384 clones were randomly from a protein expression The were by PCR using a single primer pair a T7 a binding and a T7 was on the chip using the unpurified PCR products as of the expression detection by an at the in which template was to the signals in the The of in situ expression with expression an of and clones, that the success of expression is not by the of the expression to subnanoliter volumes. may be to the of the proteins, which may or that to and subsequent A.S. cell-free for of Biotechnol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the expression to in a more of in with the which is in high throughput analysis the that that the principle of in situ expression on a microarray can be to a of proteins and that the bottleneck of protein generation be by this we present an in situ expression of proteins on a chip as a new of the multiple spotting technique that in the detection of proteins and antibodies P. J. Lehrach H. Cahill D.J. protein microarrays: multiplex on a single Chem. 2003; PubMed Scopus Google Scholar), in the of single from P. J. G. S. Cahill D.J. Kreutzberger J. Lehrach H. J. a of antibody on Chem. 2004; PubMed Scopus Google Scholar), and the characterization of activities and P. Lehrach H. Kreutzberger J. J. on 2005; 5: PubMed Scopus Google Scholar). The in situ expression involves the spotting of DNA in a first spotting step followed by the spotting of a cell-free transcription and translation mixture in a second spotting step on top of the DNA of the in which the can allows the generation of high density protein microarrays with of to The generation of by PCR the expression of the in expression and the introduction of the PCR products can be used which interaction We and for and and for and