Functional proteomics : Generation and analysis of cDNA-encoded proteins

by Gräslund, Susanne

Recent advances in genomics have led to the accumulation of vast amounts of data about genes. However, it is the proteins and not the genes that sustain function, a fact which makes the proteins the keys to understand biology. Unlike the genome, which is a fairly constant entity, interesting biological and medical questions relate to the dynamic world of the proteomes expressed in different cell types and under different conditions. Proteomics, the large-scale analysis of proteins, now aims to identify and map entire proteomes and the challenge of studying proteins on a global scale is driving the development of new technologies for systematic analysis of protein function. The first step towards gene characterization is to obtain its complete coding sequence. Here, a method to retrieve the upstream coding sequence of a gene with a partially known downstream sequence is described. The concept is based on a polymerase-chain reaction (PCR)-assisted biotin-capture method performed directly on poly(A)+ RNA to generate a full-coding sequence of a gene. The method was applied to the gene TSG118, of which only a partial sequence was known and for which a full-length clone was not found in the available cDNA libraries. In functional analysis of proteins, information of spatiotemporal localization at cellular and subcellular level is important information, since it provides clues to function and suggests further experimentation. This thesis describes the development of systems, aimed at large-scale localization of cDNA-encoded proteins based on the generation of highly specific polyclonal antibodies. Significant efforts have been invested in the development of robust and general expression systems, suitable for high-throughput production of cDNA-encoded proteins in E. coli. A single-vector concept was first developed and evaluated for the production of 55 cDNA products from a mouse testis library. More than 90% of the expressed gene products were recovered with good yields. Subsequently, a dual vector concept was created in order to allow a more stringent procedure for affinity enrichment of the antibodies to be used for functional annotation. Antibodies generated by the described approach have been used to characterize genes encoding members of a protein complex and a tektin protein (APC/C and Tekt1). An expression system employing a novel tailor-made affinity handle was developed and evaluated. A Z-affibody, showing binding capacity toward protein A, had earlier been selected from a library constructed by combinatorial mutagenesis of a protein A domain. It was now used as an affinity handle enabling efficient recovery on protein A-Sepharose, a robust and well-documented chromatography medium. The system was used for production of cDNA-encoded proteins and in addition, two convenient affinity blotting procedures were developed to allow screening of expression efficiencies. The robustness and convenience of the presented expression system should make it suitable for various high-throughput protein expression approaches. An effort to create single vector three-frame expression systems, allowing expression of an inserted gene in three reading frames, is presented. The aim was to create a combined cloning and expression vector, in order to simplify cloning and protein expression procedures. Two vectors were constructed and although the systems would need further optimization to be used in high-throughput protein production, the principle of single vector three-frame expression was demonstrated. Key words: functional genomics, functional proteomics, gene characterization, E. coli expression, affinity purification, immunolocalization, mouse testis, spermatogenesis, rabbit antibodies, affibody, tektin, Anaphase-promoting complex or cyclosome. ISBN 91-7283-234-7 ”Den mätta dagen, den är aldrig störst. Den bästa dagen är en dag av törst.” Karin Boye