High-Throughput Protein Production and Functional Screening Although recombinant technologies are well developed, there remain many intrinsic limitati

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1 CellFree Sciences Co., Ltd. 75-1, Ono-cho, Leading Venture Plaza 201, Tsurumiku,Yokohama, JAPAN Japan TEL USA: Technology Application Note: High-Throughput Protein Production and Functional Screening

2 High-Throughput Protein Production and Functional Screening Although recombinant technologies are well developed, there remain many intrinsic limitations in their application to high-throughput (HT) protein production and functional analysis. ENDEXT Technology has the capability to overcome these limitations. As part of the technology, the newly developed Split-Primer PCR method and the HT protein synthesizer GenDecoder 1000 allow us to bypass most of the time-consuming steps. This powerful means of rapid and systematic screening can be applied to: 1. HT enzymatic testing of a large number of gene products for functional annotation 2 HT analysis of protein-protein, protein-nucleic acid, and other such interactions 3. HT protein production for their 3-D structural analysis by NMR or X-ray diffraction 4. Rapid evolutionary design of proteins

HT Protein Production and Functional Analysis Split-type PCR Plasmid cdna libraries PROTEIN SYNTHESIS Bilayer synthesis SCREENING Protein kinase ALPHA screen

400 genes / day 400 proteins / day (GenDecoder 1000) 400 targets / few hrs (combined with ALPHA screen ) 6000 proteins / 60 hrs (Super GenDecoder ) Equipment for

3 HT Protein Production and Functional Analysis Split-type PCR Plasmid cdna libraries PROTEIN SYNTHESIS Bilayer synthesis SCREENING Protein kinase ALPHA screen Nuclear receptor FCS GenDecoder 1000 Super GenDecoder Rapid Evolutionary Design of Proteins Desired proteins Vaccine candidates ALPHA screen FCS etc. 400 genes / day 400 proteins / day (GenDecoder 1000) 400 targets / few hrs (combined with ALPHA screen ) 6000 proteins / 60 hrs (Super GenDecoder ) Equipment for ALPHA screen and FCS analysis is commercially available from PerkinElmer and Olympus, respectively. ALPHA screen is a trademark of PerkinElmer. 400 targets / day (combined with FCS)

4 Split-Primer PCR - a breakthrough technology for high-throughput protein expression One of the bottlenecks in high-throughput (HT) protein synthesis is the laborious cloning step to generate the template for transcription. The advent of the PCR method has made possible the direct use of replicated cdnas for transcription without purification. In a conventional PCR method that uses four primers, the complete SP6 promoter sequence is used for one of the four primers. As a result, the amplified templates are mostly short cdnas, which result in short mrnas to produce small peptides. This is possibly caused by the generation of primer-dimer artifacts (see Conventional PCR: e and f). Prof. Endo and colleagues designed a set of primers, in which the SP6 promoter sequence is split into two primers. The result is that the complete sequence is repllicated only when the two split sequences are joined correctly. This primer design has made it possible to eliminate short mrnas and produce only complete mrnas and translation products. (see Split-type Primer method for HT construction of the DNA Templates: e and f ). This Split-Primer PCR method is a powerful tool for HT protein production. For more information, please see the paper titled A cell-free protein synthesis system for highthroughput proteomics. ( Tatsuya Sawasaki, Tomio Ogasawara, Ryo Morishita, and Yaeta Endo Proceedings of National Academy of Science USA, November 12, 2002 vol. 99 no. 2)

5 Split-type Primer method for HT-construction of the DNA templates Conventional PCR This figure is by courtesy of Dr. Sawasaki and Prof. Endo, Ehime Univ.

6 Screening of 530 Protein Kinases Phosphating a CaMKII Peptide This figure is by courtesy of Dr. Sawasaki and Prof. Endo, Ehime Univ. SAM2 Biotin is a registered trademark of Promega.

7 Screening of 530 Protein Kinases Phosphating a CaMKII Peptide This slide shows an example of high-throughput (HT) protein production and functional analysis using ENDEXT Technology. More than 500 kinases derived from Arabidopsis (plant) were expressed comprehensively. These kinases were mixed with a biotinylated-synthetic peptide specific for calmodulin-dependent protein kinases (CaMKII). Each kinase was mixed with this target peptide and then incubated with gannma- 32 P-ATP. After incubation, the peptide was captured on a membrane and its radioactivity was measured. Seven kinases specifically phosphorylated the target. The entire bulk of this experiment was completed in 2 days.

8 High-throughput Identification of Kinase Substrates (ex. CaMKIId Substrates) Protein kinase assay using ALPHA screen Protein A conjugated Acceptor Beads 680 nm *0 2 Streptavidin-coated Donor Beads biotin bls P Protein anti-phosphoser/thr monoclonal antibody Substrates: Proteins highly expressed in cancer cell This figure is by courtesy of Dr. Sawasaki and Prof. Endo, Ehime Univ. Equipment for ALPHA screen is commercially available from PerkinElmer. ALPHA screen is a trademark of PerkinElmer.

9 High-Throughput Identification of Kinase Substrates (ex. CaMKIId Substrates) Protein kinases play a crucial role in cell growth, especially the growth of cancer cells. Therefore, the identification of potential kinase targets (substrates) is important for drug discovery. We selected 100 proteins that are highly expressed in caner cells, and they were synthesized in biotinylated form by ENDEXT Technology (patent pending). These biotinylated proteins were assessed for phosphorylation by calmodulin-dependent kinase II delta (CaMKIId) using AlphaScreen (PerkinElmer) in a high-throughput manner. CFS offers contract R&D and/or collaborative R&D services to identify kinase substrates.

10 Screening Nuclear Receptors using Fluorescence Correlation Spectra (FCS) '("" ''"" '""" &"" %"" $"" #""!"" )*+,- DNA *.'+' *.'+(// *.'0(/// *.'01/// *.'2'/// *.'2(/// *.'21/// *.')'/// *.')(/// *.'3'/// *.'3(/// *.'31/// *.'4(/// *.'41/// *.'45/// *.'6'//// *.'6(//// *.'61//// *.(+'/// *.(+1/// *.(0'/// *.(01/// *.(2(/// *.(7'/// *.(71/// *.(3'/// *.(3(/// *.(3#/// *.1+'/ *.10'// *.10(/// *.101/// *.12'/// *.5+'/// *.!+'/// *.!+(/// Nuclear receptors synthesized in wheat germ cell-free protein synthesis system This figure is by courtesy of Dr. Sawasaki and Prof. Endo, Ehime Univ.

11 Nuclear receptors are important targets for drug discovery. In this experiment, 36 human nuclear receptors were synthesized by ENDEXT Technology and analyzed for interaction to putative common target DNA by Fluorescence Correlation Spectroscopy (FCS) ( patent pending). Out of 36, 11 nuclear receptors (yellow bar) showed interaction to the target DNA. Using ENDEXT Technology, these experiments could be performed in a high-throughput manner. CFS offer the contract R&D and/or collaborative R&D services for analyzing interaction between nuclear receptors and target DNAs.

Protein Modification by Molecular Evolution Examples: Heat stability, high affinity, high enzymatic activity, etc. Target Gene Introduction of mutation by error prone PCR, domain shuffling, etc.

12 Protein Modification by Molecular Evolution Examples: Heat stability, high affinity, high enzymatic activity, etc. Target Gene Introduction of mutation by error prone PCR, domain shuffling, etc. Mutated Gene Mutated Protein Desired Protein with Genetic Information 1. HT protein expression by wheat germ system 2. Genotype-phenotype linkage (ribosome display, etc.) Screening by function

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