1. Cell-free Bacterial Yeast Insect Mammalian Protein Expression Systems

2. Protein Expression in Bacteria 1.Advantages/disadvantages 2.Genetic elements essential for the expression 3.Cloning strategies 4.Overview of the available expression systems and expression strains 5.Design of cloning procedures using the VNTI program

3. Advantages Fast growth Cheap medium and equipment for growing Good knowledge of the host

4. Disadvantages Limitation for expression of eukaryotic proteins due to: different frequencies with which the different codons appear in genes of these organisms E.g. CGT, CGC, CGG, AGG, AGA, CGA code for arginine, but the last 3 (AGG, AGA, CGA) are rarely used in E. coli and it has low amounts of respective tRNAs. differences in post-translational modifications (SS bonds, glycosylation etc)SS bonds, glycosylation

5. Disadvantages Accumulation of lipopolysaccharides (generally referred to as endotoxins) …

6. Goals To obtain as much as possible /good expression+good cell growth soluble folded protein /reduced aggregation in a form that is easy to purify /use of secretion and tags Common problem: High expression=danger of aggregation, decreased cell growth

7. Genetic Elements Essential for Expression

8. RBS, START, and STOP RBS RBS 5-9 n START GAAGGAATTCAGGAGCCCTTCACCATG...... Ribosome Bindind Site (RBS): START codons: E. coli uses 77% ATG (AUG), 14% GTG (GUG), 8% TTG (UUG) and a few others STOP codons: TAG (UAG), TGA (UGA), TAA (UAA)

9. Genetic Elements Essential for Expression

10. Promoters Host’s promoters 2500 in the entire genome of E. coli K12 strain Most frequently used: Plac / Ptac / Ptrc, P PBAD, rha PBAD -Regulation of expression Promoters from phages T7, T3, SP6, T5, P L - Highly efficient and specific expression

11. Plac: Regulation

12. Plac, Ptac, Ptrc: Characteristics Level of expression (inductor) Key features PlacLow level up to middle (IPTG) Weak, regulated. Suitable for expression of gene products at very low intracellular level. Comparatively expensive induction. Ptac Ptrc (trp-lac) Moderately high (IPTG) High-level, but lower than T7 system. Regulated expression still possible.Comparatively expensive induction. High basal level.

13. P PBAD : Regulation

14. P PBAD and Rha PBAD Level of expression (inductor) Key features P PBAD Variable from low to high level (L-arabinose) Can fine-tune expression levels in a dose-dependent manner. Tight regulation possible. Low basal level. Inexpensive inducer. rha PBAD Variable from low to high level (L-rhamnose) Tight regulation. Low basal activity. Relatively expensive inducer.

15. Phage Promoters Level of expression (inductor) Key features T7 T5 Very high High Utilizes T7 RNA polymerase. Utilizes E. coli RNA polymerase. PLPL Moderately high (temperature shift) Temperature-sensitive host required. Less likelihood of "leaky" un-induced expression. Basal level; high basal level by temperatures below 30°C. No inducer.

16. Combinations

17. Genetic Elements Essential for Expression

18. Replication Origin PlasmidRepliconCopy Number pBR322pMB115-20 pUC 500-700 pACYCp15A18-22 pSC101 5 colE1 15-20

19. Co-expression from two plasmids

20. Protein Expression in Bacteria Part2 1.Cloning strategies 2.Overview of the available expression systems and expression strains 3.Design of cloning procedures using the VNTI program

21. Types of Expression Vectors 123123

22. Insertion into Transcriptional Vectors

23. Insertion into Translational Vectors

24. Cloning Using Restriction Enzymes NcoI HindIII

25. Cloning Using A-overhangs

26. TA-Cloning with Topoisomerase

27. Directional Cloning CACC

28. Gateway Technology

29. Expression of Fusion Proteins We may fuse the target protein with various tags to facilitate its purification or detection HHHHHH-target, epitope-target highly soluble proteins to improve solubility and to facilitate purification Thioredoxin-target, GST-target signal peptides or other proteins or domains to promote secretion SP-target

30. ‘Short’ Fusion Protein Construction ATG CAT CAC CAT CAC CAT CAC

31. ‘Long’ Fusion Protein Construction NcoIHindIII PstI

32. pUC18/19 Transcriptional vector

33. pTrc99 Translational vector

34. pQE Translational vector + CDR

35. pET

36. pCR&pEXP

37. pBAD

38. Expression strains

39. Expression optimization To optimase: Level of inducer (e.g. arabinose) Time of induction Temperature of the induction step (popular - 18 o C overnight)