Part II: Biology

Chemical Biology, WS

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Markus Müller (Dipl. Biologe)• Studium 1994-1999 Biologie in Marburg

• Doktorarbeit (Pflanzenphysiologie) 1999-2003 in Marburg

• Postdoc 2003-2006 in Umeå, Schweden

• Seit 2006 angestellter akademischer Rat in München

Diploma thesis (1999)

PhD thesis (2004)

Postdoc (2006)

Carell-group

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

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.

Definitions

„Chemical biology may be defined as the developmentand use of chemistry techniques for the study ofbiological phenomena“ (Breinbauer & Waldmann)

Examples

Structural X-linking Mass Spectrometry

Activity-based protein profiling

Next/Third Generation Sequencing

Therapeutic proteins

Novel therapeutics, e.g. siRNA

K Murakami et al. Science 2013;science.1238724Published by AAAS

Structural Proteomics

Fig. 1 Scheme for protein chemical modifications.

Aerin Yang et al. Science 2016;science.aah4428

Published by AAAS

Activity‐based protein profiling

Third‐generation sequencing

Pharmaceutical conjugation

siRNA delivery

The Basics of Cloning & Protein Production

Initial Considerations

Choice of Expression System• Bacteria

• Yeast

• Insect Cells

• Mammalian Cells

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 geneticmaterial• 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 tohumans, animals or plants).

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

Safety Considerations

Genetic engineering, where according to current scientificknowledge, no risk to human health and the environmentexists. (no pathogens!)

Genetic engineering, where according to current scientificknowledge, medium risk to human health and the environmentexists. (accute pathogens, but reduced transmission)

Genetic engineering, where according to current scientificknowledge, little risk to human health and the environmentexists. (facultative pathogens allowed)

Genetic engineering, where according to current scientificknowledge, high risk to human health and the environmentexists. (pathogens transmittable e.g. by air)

S1

S2

S3

S4All safety levels above one create a significantly higher work load and shouldbe avoided whenever possible.

Initial Considerations

Purification Strategy• Native purification

• Affinity tags• His

• Strep

• GST/MBP/Trx

• Epitope

• Thermal Stability

Initial Considerations

Molecular Cloning• Classic (restriction digest)

• Ligation independent cloning

• Recombination (Gateway, Stargate)

• Gene synthesis

Bacterial Systems

• Escherichia coli• Bacillus subtilis• Caulobacter crescentus• Lactococcus lactis

Advantages:• Easy DNA transfer• Easy growth (cheap!)• Simple and small promoters• High yieldDisadvantages:• No posttranslational

modification• Folding of large proteins

difficult• Endotoxins

e.g. Bacillus strains

e.g. E. coli strains

Eukaryotic Systems

Nucleus

Mitochondrium

ER

Golgi• Yeasts• Insect cells• Mammalian cells• Plants

Advantages:• Human-like posttranslational

modifications• Enhanced folding of large

proteins• Enhanced secretionNachteile:• Lower yields• Expensive growth media• More complex handling• Difficult to transfect (smaller

choice of vectors)

Glycosylation patterns

Bioinformatics

http://www.ncbi.nlm.nih.gov/gene

Choice of Source

www.uniprot.org

Learn from structure

Get the nucleotide sequence

Codon Usage

E.coli

H.sapiens

Summary• Expression host

• Purification strategy

• Biosafety considerations

• Source of genetic material

• Cloning strategy

• Know your gene/protein