Search: onr:"swepub:oai:DiVA.org:kth-29" >
Protein production,...
Protein production, characterization and structure determination in structural genomics
-
- Woestenenk, Esmeralda A., 1975- (author)
- KTH,Bioteknologi
-
Härd, Torleif (thesis advisor)
-
- Karlsson, Göran, Doc (opponent)
- Charlmes Tekniska Högskolan, Göteborg
-
(creator_code:org_t)
- ISBN 9172838388
- Bioteknologi, 2004
- English.
- Related links:
-
https://kth.diva-por... (primary) (Raw object)
-
show more...
-
https://urn.kb.se/re...
-
show less...
Abstract
Subject headings
Close
- This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy. The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days. The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus. Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein
Subject headings
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Keyword
- Biology
- protein production
- protein solubility
- fusion tags
- nuclear magnetic resonance
- structure determination
- Biologi
- Biology
- Biologi
Publication and Content Type
- vet (subject category)
- dok (subject category)
Find in a library
To the university's database