1 General consideration about Gene Expression and expression studies Expression studies Expression Host -> Expression System Promoter system -> expression vector Properties of product -> stability Production level

2 Expression studies 1. Analyzing Transcription - Northern blot - Micro array - real-time PCR - Primer extension 2. Promoter studies Use of report genes to study regulatory elements 3. Analyzing Translation - Western blot - immuno assays - 2D electrophoresis - proteomics

3 Northern Blot -> to study transcription level

4 Studying Transcription Microarray technique – DNA chips

5 Studying Transcription Microarray technique – DNA chips

6 Microarray technique to study upregulation and downregulation of gene expression

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8 Studying Transcription Primer Extension

9 Promoter Studies Used reporter genes: - Lac Z - GFP - Luciferase PromoterReporter gene

10 Promoter studies by using reporter genes

11 Use of green fluorescent protein (GFP) as a reporter gene. Page 119

12 Analyzing Translation – Western Blot

13 2 D Electrophoresis

14 Comparison of expression systems

15 Prokaryotic Expression vector

16 Eukaryotic Expression vector

17 -> mainly in prokaryotes (E. coli) -> 2µ plasmid in yeast -> some virus vectors in mammalien cells -> mainly in eukaryotes -> some bacterial systems (Bacillus)

18 Gene Expression Transcriptional start Translational start

19 Principal factors in bacterial expression

20 Type of expression vectors

21 Prokaryotic Translational vector PstI T7/la c phoAcutinase NarI SalI HindIII BamHI phoA-cut NdeI S/D Term pFCEX1

22 Gene Expression Gene copy number: 1. Plasmid copy number: The copy-number of a plasmid in the cell is determined by regulating the initiation of plasmid replication. The initiation of plasmid replication may be controlled by: - the amount of available primer (RNA) - the amount of essential replication proteins - the function of essential replication proteins. 2. Gene dosage -> number of genes integrated into chromosome - prokaryotic systems -> i.e. Transposons, phages, recombinantion - mainly eukaryotic systems

23 Incompatibility of plasmids: Not all plasmids are able to coexist in the same cell. Plasmids which have the same replication control functions are incompatible, and are assigned to the same incompatibility group (inc group). Plasmids of one incompatibility group are related to each other, but cannot survive together in the same bacterial cell, as only different kinds of plasmids are compatible. Ensures that we can make libraries -> just one plasmid taken up by one cell

24 Homologous integration into chromosome Insertion on Bacillus subtilis chromosome

25 mRNA stability

26 Translation Initiation

27 Codon Usage of Organisms Rare codons -> pauses translation -> lower production level

28 Fusion proteins increase production level facilitate purification (taq) detection of expression (GFP fusion) Redirection of proteins (secretion -> signal peptidases) Surface display (for screening of libraries) Tandem arrays (for small peptides, toxic proteins,..)

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30 Some problems of production in E. coli

31 Electron micrograph of an inclusion body of the protein prochymosin in an E. coli cell Page 116 Protein Folding

32 Some E.coli expression host considerations

33 Secretion of Protein -> to avoid inclusion body formation at high expression level

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