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Sample Preparation - Overview

Sample preparation for NMR and crystallization have a lot in common. Except for crystallization we may need more and for NMR the final product is labeled with 13C

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Sample Preparation - Overview

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    1. Sample Preparation - Overview by Gloria Borgstahl for the NCSB June 14, 2005

    2. Sample preparation for NMR and crystallization have a lot in common

    3. Relying heavily on this paper: Review From gene to protein: a review of new and enabling technologies for multi-parallel protein expression by Ian Hunt Novartis Horsham Research Centre, Novartis Institutes for Biomedical Research, Wimblehurst Road, Horsham, West Sussex, UK

    5. Rapid Cloning Systems

    7. Traditional Sticky End SubCloning for Expression

    9. Proteases to cleave the fusion protein make sure it wont cut your protein too

    10. Expression Systems E. coli Baculovirus-mediated insect cell expression Yeast in particular Pichia pastoris good for secreted proteins, our fermentor is suitable. Mammalian-based systems, CHO or HEK293, correct folding and post-translational modification, time consuming, laborious and expensive Cell free expression systems wheat germ extracts + plasmid or PCR product

    11. Bacterial expression with Escherichia coli Cheap, simple, quick, amenable to HT, but Eukaryotic proteins may not be modified correctly, Secretion to periplasmic space can be used to avoid the dreaded inclusion bodies that are so frequently formed by E. coli, Large complexes may be difficult T7 based pET expression plasmids Dual vectors for co-expression (complexes like tRPA, chaperones, stabilization) Cells (Origami for correct disulfides, Rosetta for human codons, etc.)

    12. Baculovirus-mediated insect cell protein expression Eukaryotic based expression system, therefore similar (but not identical) modification and processing patterns. Baculovirus-mediated system uses a helper-independent virus, propagates to high titre, suspension culture, so you can generate relatively large amounts of protein. More labor intensive and harder to streamline and adapt to HT than bacteria. New systems exist using Gateway technology that make easier to do.

    13. The End

    14. Schematic representation of: (A) BaculoDirect and (B) flashBac recombinant baculovirus generation strategies.

    16. Wave bioreactor for insect and mammalian suspension cultures

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