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FU Berlin
Digitale Dissertation

Konrad Büssow :
Arrayed cDNA libraries for antibody screening and systematic analysis of expression products
Geordnete cDNA Banken für Antikörper-Screening und systematische Analyse von Expressionsprodukten

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Abstract

Functional analysis of the proteins expressed in a specific tissue and/or stage of development is an essential step towards understanding biological processes. No technique has yet been available to go directly from sequence information on individual cDNA clones to the protein products. In the approach described here, arrayed cDNA expression libraries from human fetal brain (hEx1) and mouse adult kidney (mKd1) were established for the integration of DNA-based and protein-based experimental data. An E. coli plasmid vector (pQE30NST) for the expression of His6-tag fusion proteins was used to clone cDNA synthesised by oligo(dT) priming. Using automated systems, 27,600 clones of the mKd1 and 193,500 clones of the hEx1 library were picked, grown in microtitre plates and arrayed on membrane filters at a density of 9,216 or 27,648 clones per filter. High-density DNA and protein filters were prepared and used to screen the cDNA libraries with DNA probes and antibodies in parallel. An antibody directed against the N-terminal tag sequence RGSH6 of proteins expressed from pQE30NST (RGS·His antibody, Qiagen) was used to detect expression clones. Approximately 20% of clones in the mKd1 and hEx1 cDNA libraries were detected as putative expression clones by this antibody. 37,830 putative expression clones in the hEx1 library were combined and rearrayed into new microtitre plates. A copy of this sub-library was given to the Resource Centre of the German Human Genome Project (RZPD, http://www.rzpd.de) to generate and distribute high-density DNA and protein filters. Expression products and DNA sequences of 96 randomly selected clones were analysed in detail. Protein expression and purification in microtitre plates was established. 68 out of 96 clones expressed proteins of at least 15 kd size (estimated from SDS-PAGE) which were detected by SDS-PAGE of whole cellular proteins or by metal affinity chromatography. DNA sequences derived from 5´-ends of cDNA inserts were obtained for 93 clones. 58 sequences matched human protein sequences in the SWISS-PROT and TrEMBL databases. 38 (66%) of these 58 sequences contained inserts cloned in the correct reading frame, i.e. the His6-tag and a protein coding cDNA sequence were fused in frame. 66% of sequences matched to the beginning of a protein database sequence, and the corresponding clones were therefore considered to contain complete protein-coding regions (full-length clones). Expression clones for a panel of human genes were identified in the hEx1 library. Nine cDNA probes for BMP-7, calmodulin, COX4, GAPDH, hMSH2, HSP90a, HSP90b, RXRb and VDAC1 were used for screening by DNA hybridisation. Among the positive clones, putative expression clones were selected that were also detected by the RGS·His antibody on high-density filters. Expression clones for seven genes, but not for BMP-7 and VDAC1, were found with this strategy. GAPDH and calmodulin clones comprising full protein-coding regions expressing soluble His6-tag fusion proteins were identified. Biological activity of His6-tag GAPDH and calmodulin fusion proteins was shown with enzyme assays. DNA probes and the RGS·His antibody were used in combination to detect clones expressing GAPDH and HSP90a fusion proteins. In parallel, specific antibodies directed against GAPDH and HSP90a were used to identify expression clones on high-density protein filters. All clones identified by the protein-specific antibodies expressing His6-tag GAPDH or HSP90a fusion proteins were reliably detected by the RGS·His antibody. The RGS·His detected additional clones, which either contained GAPDH or HSP90a inserts in an incorrect reading frame, or which expressed truncated proteins lacking the epitopes recognised by the protein-specific antibody. Estimated 90% of clones with inserts in an incorrect reading frame were not detected by the RGS·His antibody. A protocol for the analysis of His6-tag fusion proteins by matrix assisted laser ionisation/desorption mass spectrometry (MALDI-MS) was established. His6-fusion proteins were purified under denaturing conditions by immobilisation on Ni2+-chelate-coated magnetic beads, followed by direct digestion with trypsin. The masses of the tryptic peptides were measured and compared to masses predicted from sequences in databases. Arraying of cDNA expression libraries extends the application of DNA library arrays to protein-based screening techniques, thus creating a direct link between DNA-based and protein-based experimental data. The hEx1 expression library allows for the fast identification of expression clones for specific genes by DNA hybridisation or antibody screening. Proteins expressed from library clones can be directly used for functional assays, or, if inclusion bodies are formed, may be used as antigens to generate antibodies or possibly refolded in vitro. Libraries of expression clones displayed on high-density filters, in combination with high-throughput protein expression and purification in microtitre plates, should be a feasible approach to the construction of gene product catalogues.

Table of Contents

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Contents

1. Introduction

1.1 Expression patterns
1.2 Protein expression
1.2.1 Expression of fusion proteins
1.2.2 Expression systems
1.3 Expression libraries to study protein interactions
1.4 Arrayed DNA libraries and high-density grids
1.5 Systematic protein expression
1.6 Protein characterisation by mass spectrometry
1.7 Antibodies 9

2. Objective

3. Materials

3.1 Laboratory equipment
3.2 Chemicals, nucleotides, antibodies and enzymes
3.3 Oligonucleotides
3.4 Kits
3.5 Other materials
3.6 Buffers and media
3.7 Strains

4. Methods

4.1 Plasmid constructs
4.2 PCR and DNA sequencing
4.3 Antibody affinity purification
4.4 cDNA library construction and arraying
4.5 High-density filters for protein and DNA detection
4.6 DNA hybridisation screening of high-density filters
4.7 Antibody screening of high-density filters
4.8 Protein expression in E. coli 31
4.9 SDS-PAGE 31
4.10 Metal chelate affinity purification
4.11 Tryptic digest
4.12 Protein expression and purification in microtitre plates
4.13 Enzyme assays

5. Results

5.1 Arrayed cDNA expression libraries
5.2 Rearraying of potential expression clones in the hEx1 library
5.3 Identification of expression clones for specific genes
5.4 Characterisation of expression products by mass spectrometry

6. Discussion

6.1 Arrayed cDNA expression libraries
6.2 Rearraying of the hEx1 library
6.3 Identification of specific expression clones
6.4 Characterisation of expression products
6.5 Perspectives

7. Abstract

7.1 Abstract in English
7.2 Zusammenfassung in deutscher Sprache

8. Appendix

8.1 Abbreviations
8.2 Curriculum vitae
8.3 Publications

9. References


 
 

More Information:

Online available: http://www.diss.fu-berlin.de/1999/53/indexe.html
Language of PhDThesis: english
Keywords: expression library, cDNA array, cDNA clone, antibody, protein array
DNB-Sachgruppe: 30 Chemie
Date of disputation: 24-Mar-99
PhDThesis from: Department Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Hans Lehrach
Second Referee: Prof. Dr. Volker Erdmann
Contact (Author): buessow@molgen.mpg.de
Contact (Advisor): walter_g@molgen.mpg.de
Date created:20-Sep-99
Date available:29-Sep-99

 

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