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

2. Protein Expression in Bacteria
Advantages/disadvantages
Genetic elements essential for the expression
Overview of the available expression systems and expression strains

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:
differences in post-translational modifications
SS bonds, glycosylation etc etc
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.

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. pTrc99
Translational vector

8. Genetic Elements Essential for Expression

9. Replication Origin Plasmid Replicon Copy Number pBR322 pMB1 15-20 pUC
pACYC
p15A
18-22
pSC101 5
colE1

10. pTrc99
Translational vector

11. Genetic Elements Essential for Expression

14. Genetic Elements Essential for Expression

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

16. Genetic Elements Essential for Expression

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

18. Plac: Regulation

20. Plac, Ptac, Ptrc: Characteristics
Level of expression (inductor)
Key features
Plac
Low 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.

21. pBAD

22. PPBAD: Regulation PBAD.
- expression of the gene is controlled by the AraC activator.
Expression from PARA is induced to high levels on media containing arabinose.
Moreover, expression from PARA is tightly shut off on media containing glucose but lacking arabinose.
ranscription by PBAD
High Arabinose
Low Arabinose
High Glucose
Repressed
Low Glucose
Active

23. Level of expression (inductor)
PPBAD and RhaPBAD
Level of expression (inductor)
Key features
PPBAD
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.
rhaPBAD
(L-rhamnose)
Tight regulation. Low basal activity. Relatively expensive inducer.

24. pET

25. Level of expression (inductor)
Phage Promoters
Level of expression (inductor)
Key features T7 T5
Very high
High
Utilizes T7 RNA polymerase.
Utilizes E. coli RNA polymerase.

26. Combinations

27. Co-expression from two plasmids

28. pQE
Translational vector + CDR

29. pET

30. pCR&pEXP

31. pTrc99
Translational vector

32. Expression strains

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