The document discusses the effectiveness of deactivating repressor proteins to activate transcription in recombinant E. coli systems, emphasizing how the ratios of repressor proteins and promoter copies affect gene expression. It also covers advancements in plasmid engineering for enhanced protein production, the role of fusion proteins in protecting against degradation, and methods for purifying these proteins while ensuring they maintain their biological activity. Techniques such as using temperature-sensitive plasmids, dual plasmid systems, and affinity chromatography are highlighted as crucial for large-scale production and purification processes.

1. Effectiveness of deactivating a repressor protein and thereby activating
transcription:
• depends on the ratio of the number of repressor protein molecules to the
number of copies of the promoter sequences.
• Too many repressor molecule???
• too low repressor molecule???
Various means have been devised to keep these regulatable systems under
complete control.
1. For example, the repressor protein gene and the promoter that it regulates
may be placed on two different plasmids that maintain different numbers of
copies per cell; this arrangement maintains the appropriate ratio between
the repressor protein and the promoter. Usually, the repressor gene is
placed on a low-copy-number plasmid that maintains about 1 to 8 copies
per cell, and the cloned gene with its promoter sequence is inserted into a
high-copy-number plasmid that maintains about 30 to 100 copies per cell.
2. Alternatively, the repressor protein gene may be carried as a single gene in
the chromosomal DNA, an arrangement that keeps repressor protein levels
low.
3. In systems that use the lac promoter, a mutant form of the lacI gene (lacIq)
that produces much higher levels of the lac repressor is often used to
decrease transcriptional leakiness under noninduced conditions, i.e.,
transcription of a cloned gene in the absence of inducer.

2. Increasing Protein Production:
• Plasmid pCP3 was created in an effort to obtain the highest possible level of
foreign-protein production in a recombinant E. coli strain.
• This plasmid contains the strong pL promoter, the β-lactamase gene
(ampicillin resistance gene) as a selectable marker, a multiple cloning
sequence immediately downstream from the promoter, and a temperature-
sensitive origin of DNA replication that increases the plasmid’s copy
number 5- to 10-fold when the growth temperature is increased to 42°C .
• E. coli cells that carry the plasmid pCP3 are first grown at 28°C and then
shifted to 42°C. At the lower temperature, the cI repressor, which is
integrated into the host E. coli chromosomal DNA, is functional, the pL
promoter is turned off, and the plasmid copy number is normal (about 60
copies per cell).
• At the higher temperature, the temperature-sensitive cI repressor is
inactivated, the pL promoter is active, and the plasmid copy number
increases to around 600 copies per cell.
• These properties make pCP3 a particularly effective expression vector. When
the gene for the enzyme T4 DNA ligase is inserted into the multiple cloning
site of pCP3, about 20% of the cellular protein produced at 42°C is T4 DNA
ligase.

4. Large-Scale Systems:
• In small culture vessels (1 to 5 liters), induction is readily achieved either by
shifting the temperature or by adding a chemical inducer.
• In pilot plant-size (20 to 200 liters) and industrial-size (>200 liters)
bioreactors, however, a shift in temperature requires time (30 to 60 minutes)
and energy, both of which can be costly.
• Similarly, the cost of a chemical inducer, such as IPTG, that is required for
the expression of a cloned gene in a large-scale bioreactor can make the
overall process uneconomical.
• To overcome some of the problems associated with the use of the pL
promoter for large-scale fermentations, a two-plasmid system has been
developed.
• The cI repressor was placed under the control of the trp promoter and
inserted into a low-copy-number plasmid. The use of a low-copy-number
plasmid ensures that excess cI repressor molecules are not produced.
• A second plasmid carries a cloned gene under the control of the pL
promoter.
• The trp promoter is turned on in the absence of tryptophan, so the cI
repressor protein is synthesized and the pL promoter is turned off. In
contrast, the trp promoter is turned off in the presence of tryptophan, so the
cI repressor protein is not synthesized and the pL promoter is fully active.
• With this two-plasmid system, cells can be grown on an inexpensive
medium consisting of molasses and casein hydrolysate, which contains
only very small amounts of free tryptophan, and then induced to express the

5. • In trial runs of this
system, cloned β-
galactosidase and
citrate synthase genes,
after induction by
addition of tryptone to
the medium,
represented 21 and 24%
of the cellular protein,
respectively.

6. Fusion Proteins
• Often, foreign proteins, especially small ones, occur in minute quantities
when they are produced in heterologous host cells.
• This apparently low level of expression is, in many instances, actually due to
degradation of the foreign protein.
• One way to solve this problem is to engineer a DNA construct that encodes a
target protein that is in frame with a stable host protein.
• This combined, single protein, which is called a fusion protein, protects the
cloned gene product from attack by host cell proteases.
• In a number of studies, proteins synthesized from cloned genes have been
found to be resistant to degradation when they are part of a fusion protein,
whereas when they are expressed as separate proteins, they are susceptible
to degradation by proteolytic enzymes (proteolysis).
• Fusion proteins are constructed at the DNA level by ligating a portion of the
coding regions of two or more genes.
• Knowledge of the nucleotide sequences of the various coding segments that
are joined at the DNA level is essential to ensure that the ligation product
maintains the correct reading frame.
• If the combined DNA has an altered reading frame, i.e., a sequence of
successive codons that yields either an incomplete or an incorrect
translation product, then a functional version of the protein encoded by the
cloned target gene will not be produced.

7. Cleavage of Fusion Proteins
• It is undesirable to produce a fusion protein as the final product.
• the presence of the host protein segment makes most fusion proteins
unsuitable for clinical use and may affect the biological functioning of the
target protein.
• fusion proteins require more extensive testing before being approved by
regulatory agencies, such as the U.S. Food and Drug Administration.
• strategies have been developed to remove the unwanted amino acid
sequence from the target protein.
• One way to do this is to join the gene for the target protein to all or a portion
of the gene for another protein (the stabilizing fusion partner) with
oligonucleotides that encode short stretches of amino acids that are
recognized by a specific nonbacterial protease.
• For example, an oligonucleotide linker encoding the amino acid sequence
Ile-Glu-Gly-Arg can be joined to the cloned gene.
• Following synthesis and purification of the fusion protein, a blood
coagulation factor called Xa can be used to release the target protein from
the fusion partner, because factor Xa is a specific protease that cleaves
peptide bonds uniquely on the C-terminal side of the Ile-Glu-Gly-Arg
sequence.
• Moreover, because this peptide sequence occurs rather infrequently in
native proteins, this approach can be used to recover many different cloned

8. Uses of Fusion Proteins:
• a specific antigenic site that is required in large amounts and is part of a
fusion protein may be used for research or diagnostic purposes as long as
the stabilizing protein does not interfere with the correct folding of the
antigenic site.
• In this case, the fusion protein can be used as an antigen, and any
antibodies that are directed against the stabilizing protein can be removed
by absorption with this protein alone, thus leaving in the antiserum only
those antibodies that bind to the targeted protein sequence.

9. • a fusion cloning vector that included
the 5′-terminal segment of the E. coli ompF gene, which directs the
synthesis of an outer membrane protein, and
a portion of the E. coli lacZ (β-galactosidase) gene was constructed and
used to generate antibodies against selected target proteins.
• The ompF gene segment contributed the signals for the initiation of both
transcription and translation and for secretion of the fusion protein.
• Even though the truncated lacZ gene lacks the codons for the first 8 amino
acids, the shortened protein encoded by this gene fragment is still
enzymatically active. This form of the enzyme β-galactosidase is able to
function with almost any peptide fused to its N terminus.
• The lacZ gene was cloned on the vector at a location that put it in an altered
reading frame with respect to the ompF leader sequence. Therefore, no
functional β-galactosidase was produced.
• However, any cloned target DNA that had both ompF and lacZ in frame
would produce a three-part hybrid protein that comprised a portion of the
OmpF amino acid sequence, the protein encoded by the cloned target gene,
and the functional C-terminal portion of β-galactosidase, whose activity is
readily visualized on plates.
• Such a hybrid protein can be used either as an antigen to produce antibodies
that will cross-react with the protein encoded by the cloned gene or as a

11. • fusion proteins simplify the purification of recombinant proteins. This
approach is useful for purification of proteins expressed in either
prokaryotic or eukaryotic host organisms.
• For example, a vector that contains the human interleukin-2 gene joined to
DNA encoding the marker peptide sequence Asp-Tyr-Lys-Asp-Asp-Asp-Asp-
Lys has the dual function of reducing the degradation of the expressed
interleukin-2 gene product and then enabling the product to be purified.
• Interleukin-2 is a biological factor that stimulates both T-cell growth and B-
cell antibody synthesis.
• Following expression of this construct, the secreted fusion protein can be
purified in a single step by immunoaffinity chromatography, in which
monoclonal antibodies against the marker peptide have been immobilized
on a polypropylene (or other solid) support and act as ligands to bind the
fusion protein.

12. • Because the marker peptide is relatively small, it
does not significantly decrease the amount of host
cell resources that are available for the production
of interleukin-2; thus, the yield of interleukin-2 is not
affected by the concomitant synthesis of the marker
peptide.
• In addition, while the fusion protein has the same
biological activity as native interleukin-2, to satisfy
the government agencies that regulate the use of
pharmaceuticals, it is still necessary to remove the
marker peptide if the product is to be used for
human immunotherapy or other medical purposes.
• In this system, the marker sequence may be
specifically removed by treatment of the fusion
protein with bovine intestinal enterokinase (which
is a highly specific protease, despite its name).

13. • In many instances antigen-antibody complexes that form during the
immunoaffinity process are difficult to separate without the use of
denaturing chemicals.
• As an alternative, it has become very popular to generate a fusion protein
containing six or eight histidine residues attached to either the N- or C-
terminal end of the target protein.
• The histidine-tagged protein, along with other cellular proteins, is then
passed over an affinity column of nickel–nitrilotriacetic acid.
• The histidine-tagged protein, but not the other cellular proteins, binds
tightly to the column.
• The bound protein is eventually eluted from the column by the addition of
imidazole (the side chain of histidine).
• With this protocol, some cloned and overexpressed proteins have been
purified up to 100-fold with greater than 90% recovery in a single step.
• In addition, this system can be utilized to purify denatured proteins, for
example, following solubilization of inclusion bodies and before the
solubilized proteins are renatured.