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 β-galactosidase) (Table 6.4) Met, Ser, Ala at : 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.
Facilitating Protein Folding Inclusion body Insoluble aggregates of expressed protein The in vivo insolubility of proteins is often a consequence of their incorrect folding. Various strategies to prevent inclusion body formation Tagging with other proteins (Fig. 6.21) Thioredoxin, GST Leader sequence is used to facilitate the secretion to the periplasm. Overexpression of disulfide bond-forming proteins (DsbA, DsbB, DsbC, DsbD). (Fig. 6.22)
Regulation of the synthesis of a thioredoxintarget protein fusion without tryptophan with tryptophan Enterokinase cleavage site
Overexpression of disulfide bond-forming protein (DsbC)
Coexpression Strategies Cultivation at low temperatures Beneficial to proper protein folding However, host cell growth is slow at low temperature Expression of the chaperonin 60 gene (cpn60) and the cochaperonin 10 gene (cpn10) obtained from psychrophilic bacterium E. coli host gained the ability to grow at a high rate at low temperatures (4 to 10 o C) The expression of the temperature-sensitive esterase (target protein) increased 180-fold.
Overcoming Oxygen Limitation Oxygen limitation Slow growth Enter stationary phase Protease production Use of protease-deficient host strains Proteases are also important for the degradation of abnormal or defective proteins, which is a house keeping function for cell viability. Mutation in both rpoh and degp decreased the protein degradation of secreted proteins. rpoh (RNA polymerase sigma factor that is responsible for heat shock protein synthesis) degp (protease required for cell growth at high temperatures)
Overcoming Oxygen Limitation 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
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 6.25, Figure 6.26) 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
DNA Integration into the Host Chromosome Double cross over Single cross over
Multiple Integration Expression of α-amylase in B. subtilis Integration of plasmid with α-amylase gene and chloramphenicol resistance marker on B. subtilis chromosome Isolation of clones with multiple integration by selection under high chloramphenicol Copies/genome α-amylase activity (U/ml) 2 500 5 2,300 7 3,100 9 4,400 Multicopy plasmid 700
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.
Removing Selectable Marker Genes The integration of a selectable marker gene along with a gen of interest is helpful for identifying transformed cells under laboratory conditions. However, the presence of a selectable marker gene for antibiotic resistance in a genetically modified organism that is released into the environment is not desirable.
Removing Selectable Marker Genes Cre-loxP recombinantion system Cre recombinase loxp sites: two 34-bp recombination sites Removal of a marker flanked by loxp sites by expression of Cre enzyme (on a separate plasmid under the control of lac promoter)
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
Secretion into the Periplasm
However, the presence of a signal peptide sequence (e.g. MBP signal) does not guarantee a high rate of secretion.
Increasing Secretion In may cases, secretion of heterologous proteins in E. coli is dependent on the translational level of the protein. Translational overload Inhibition of protein secretion If secretion is important, one way may be to lower the level of expression.
Secretion into the medium E. coli and other gram-negative microorganisms generally cannot secrete proteins into the medium because of the presence of an outer membrane. Use of gram-positive prokaryotes or eukaryotic cells as a host cell Use of genetic manipulation to engineer gram negative bacteria
Secretion into the medium (1) Secretion to the periplasm using a secretion signal (2) 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
Secretion into the medium Secretion of E. coli protein to the medium is quite rare. The small protein YebF is naturally secreted to the medium without lysing the cells or permeabilizing the membranes. Target proteins are fused to the YebF.
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
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
Prevention of Metabolic Load Prevention of metabolic load Low copy number plasmid for expression Integration of DNA into host chromosome Inducible promoter Codon usage Optimization for the maximum yield Protein expression levels One way to increase the production of target protein is to accept a modest level of foreign-gene expression (perhaps 5% of total cell protein). Instead focus on attaining a high cell density