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High-Throughput Evaluation of Protein Folding Conditions and Expression Constructs for Structural Genomics

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Scheich,  Christoph
Max Planck Society;

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Scheich, C. (2004). High-Throughput Evaluation of Protein Folding Conditions and Expression Constructs for Structural Genomics. PhD Thesis, Universität, Potsdam.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0010-87E5-4
Abstract
For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 g protein i s used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 g) immobilised on metal chelate resins.