Chemical Biology, WS. Part II: Biology

Transcription

1 Chemical Biology, WS Part II: Biology

2 1

3 Markus Müller (Dipl. Biologe) Studium Biologie in Marburg Doktorarbeit (Pflanzenphysiologie) in Marburg Postdoc in Umeå, Schweden Seit 2006 angestellter akademischer Rat in München

4 Diploma thesis (1999)

5 PhD thesis (2004)

6 Postdoc (2006)

7 Carell-group

8 Literatur C. Mühlhardt: Der Experimentator - Molekularbiologie/Genomics H. Rehm & T. Letzel: Der Experimentator: Proteinbiochemie/Proteomics Lottspeich/Engels/Zettlmeier Lay: Bioanalytik H. Waldmann & P. Janning: Chemical Biology Andrew Miller & Julian Tanner: Essentials Of Chemical Biology: Structure and Dynamics of Biological Macromolecules

9 Definitions Biochemistry The chemistry of biological systems: study of enzyme action. Biological chemistry (Bio-organic and Bio-inorganic Chemistry) The synthesis of bio-molecules, both natural and model compounds: e.g. DNA, sugar, lipid and peptide synthesis. Chemical biology The study and manipulation of biological systems by chemical (synthetic) means. Synthetic Biology Building and re-building of bio-inspired structures to even artificial lifeforms.

10 Definitions Chemical biology may be defined as the development and use of chemistry techniques for the study of biological phenomena (Breinbauer & Waldmann)

11 Examples Structural X-linking Mass Spectrometry Activity-based protein profiling Next/Third Generation Sequencing Therapeutic proteins Novel therapeutics, e.g. sirna

12 Structural Proteomics Published by AAAS K Murakami et al. Science 2013;science

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14 Fig. 1 Scheme for protein chemical modifications. Aerin Yang et al. Science 2016;science.aah4428 Published by AAAS

15 Activity based protein profiling

16 Third generation sequencing

17 Pharmaceutical conjugation

18 sirna delivery

19 The Basics of Cloning & Protein Production

20 Initial Considerations Choice of Expression System Bacteria Yeast Insect Cells Mammalian Cells

21 Glossary Genetic engineering comprises: Generation of genetically modified organisms (GMO s) Use, cultivation, storage, inactivation and disposal as well as transport Organism: every biological entity able to proliferate or transfer genetic material Living organisms Living parts of organisms (e.g. single cells) Spores Viruses GMO: genetically modified organism (dt.: GVO) Safety Level 1: All organisms that, by current scientific knowledge, do not pose any risk to human health and the environment (non-pathogenic to humans, animals or plants).

22 Non-genetic engineering: In-vitro fertilization Natural processes like conjugation, transduction, transformation Chemical or physical mutagenesis Use of purified DNA or protein extracts (even if originating from GMO s) except GMO s are being used as donor or acceptor organisms

23 Safety Considerations S1 S2 S3 S4 Genetic engineering, where according to current scientific knowledge, no risk to human health and the environment exists. (no pathogens!) Genetic engineering, where according to current scientific knowledge, little risk to human health and the environment exists. (facultative pathogens allowed) Genetic engineering, where according to current scientific knowledge, medium risk to human health and the environment exists. (accute pathogens, but reduced transmission) Genetic engineering, where according to current scientific knowledge, high risk to human health and the environment exists. (pathogens transmittable e.g. by air) All safety levels above one create a significantly higher work load and should be avoided whenever possible.

24 Initial Considerations Purification Strategy Native purification Affinity tags His Strep GST/MBP/Trx Epitope Thermal Stability

25 Initial Considerations Molecular Cloning Classic (restriction digest) Ligation independent cloning Recombination (Gateway, Stargate) Gene synthesis

26 Bacterial Systems Escherichia coli Bacillus subtilis Caulobacter crescentus Lactococcus lactis Advantages: Easy DNA transfer Easy growth (cheap!) Simple and small promoters High yield Disadvantages: No posttranslational modification Folding of large proteins difficult Endotoxins

27 e.g. Bacillus strains e.g. E. coli strains

28 Eukaryotic Systems Golgi Nucleus ER Yeasts Insect cells Mammalian cells Plants Mitochondrium Advantages: Human-like posttranslational modifications Enhanced folding of large proteins Enhanced secretion Nachteile: Lower yields Expensive growth media More complex handling Difficult to transfect (smaller choice of vectors)

29 Glycosylation patterns

30 Bioinformatics

31 Choice of Source

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33 Learn from structure

34 Get the nucleotide sequence

35 Codon Usage E.coli H.sapiens

36 Summary Expression host Purification strategy Biosafety considerations Source of genetic material Cloning strategy Know your gene/protein