Functional proteomics : Generation and analysis of cDNA-encoded proteins
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.
”Den mätta dagen, den är aldrig störst.
Den bästa dagen är en dag av törst.”
School:Kungliga Tekniska högskolan
Source Type:Doctoral Dissertation
Keywords:functional genomics; functional proteomics; gene characterization; E. coli expression; affinity purification; immunolocalization; mouse testis; spermatogenesis; rabbit antibodies; affibody; tektin; Anaphase-promoting complex or cyclosome.
Date of Publication:01/01/2002