1. Increasing Protein Stability
Stability depends on
the extent of disulfide bond
the presence of certain amino acids at the N terminus
(Amino acids added to N terminus of b-galactosidase)
(Table 6.4)
Met, Ser, Ala : half-life > 20 h
Arg: half-life = ~ 2 min
Internal PEST sequence
rich in proline (P), glutamic acid (E), serine (S), and threonine (T)
make a protein more susceptible to proteolytic degradation
The stability would be enhanced if its PEST regions could be altered by genetic manipulation.

2. Protein Folding Inclusion body
Insoluble aggregates of expressed protein
Strategies to prevent inclusion body formation
Tagging with other proteins
Thioredoxin, GST
Leader sequence is used to facilitate the secretion to the periplasm.
Overexpression of disulfide bond-forming protein (DsbC)

3. Overcoming Oxygen Limitation
Slow growth
Enter stationary phase
Protease production
Use of protease-deficient host strains
Proteases are also required for cell viability.
rpoH (heat shock sigma factor) and degP (protease for high temperature growth) mutants
Decrease in protein degradation of secreted proteins
Bacterial hemoglobin
Hemoglobin-like molecule in Vitreoscilla bacterium
It binds oxygen from the environment and increase the level of available oxygen inside cells.
Expression in E. coli to increase protein synthesis

4. DNA Integration into the Host Chromosome
Plasmid ~ metabolic load
genetic instability
selective pressure (by antibiotic or essential metabolite)
Chromosomal integration
genetically stable
into a nonessential site
by a homologous recognition (Figure 6.21, Figure 6.22)
The input DNA must share some sequence similarity, usually at least 50 nucleotides, with the chromosomal DNA
Double cross over or single cross over
Use nonreplicating plasmid for integration

5. DNA Integration into the Host Chromosome
Double cross over
Single cross over

6. Multiple Integration Expression of a-amylase in B. subtilis
Integration of plasmid with a-amylase gene and chloramphenicol resistance marker on B. subtilis chromosome
Isolation of clones with multiple integration by selection under high chloramphenicol
Copies/genome a-amylase activity (U/ml)
5 2,300
7 3,100
9 4,400
Multicopy plasmid 700

7. Multiple integration at specific sites
Integration of a marker
Replacement of the marker with a target gene
To integrate additional copies of target gene, the procedure is repeated several times with different chromosomal regions

8. Removing Selectable Marker Genes
Cre-loxP
recombination system
Cre recombinase
loxP site: 34-bp recombination sites
Removal of a marker flanked by loxP site by expression of Cre enzyme (on a separate plasmid under the control of lac promoter)

9. Increasing Secretion Advantages of secretion of a target protein
No proteolytic degradation
Easy purification
Protein secretion
Addition of signal peptide (signal sequence, leader peptide)
Fusion with a secretory protein
E. coli
secretion to the periplasm for many proteins
passage through the outer membrane for a few proteins

12. Increasing Secretion Translational overload
Inhibition of protein secretion
If secretion is important, one way may be to lower the level of expression.
Permeabilization of E. coli
Inducible expression of bacteriocin release protein
The presence of bacteriocin release protein activates phospholipase A
Consequently both the inner and outer membranes are permeabilized

13. Bacteria with Increased Permeability
L-form bacteria
Bacterial lacking cell wall
Generation of L-form bacteria
Spontaneous mutations
Treatment with penicillin
Inhibition of the final step in cell wall biosynthesis
Treatment with lysozyme
Hydrolysis of saccharide linkage
High yield of protein secretion
Often stable and can continue to grow
Spheroplasts, protoplasts
Complete loss of cell wall (by enzymatic treatment)
Can grow to only a very limited extent

14. Metabolic Load
Impairment of normal cellular function of host cells by expression of foreign DNA
Replication and maintenance of high copy number plasmid
Limitation of dissolved oxygen
Depletion of certain aminoacyl-tRNAs and/or drain energy
Prevent proper localization of host proteins by foreign secretory proteins (It may “jam” export sites.)
Interference of host cell function by foreign proteins

15. Effects of a Metabolic Load
Decrease in cell growth rate
Loss of plasmid or a portion of plasmid DNA
Decrease in energy-intensive metabolic processes
Nitrogen fixation
Protein synthesis
Changes in cell size and shape
Increase in extracellular polysaccharide production
This causes the cells stick together.
Increase in translational errors

16. Prevention of Metabolic Load
Low copy number plasmid for expression
Integration of DNA into host chromosome
Inducible promoter
Optimization for the maximum yield
Protein expression levels
Cell density

17. Increasing Cell Density
Elimination of growth inhibitory waste products
e.g. prevention of acetate formation
Use glucose analogue a-glucoside to reduce glucose uptake
Use ptsG mutant (enzyme II in the glucose phosphotransferase system)
Expression of acetolactate synthase