Unit III Lecture 4

B. Tech. (Biotechnology) III Year V th Semester

EBT-501, Genetic Engineering

Unit III• Gene library: Construction cDNA library and genomic

library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity

• Marker genes: Selectable markers and Screenable markers, nonantibiotic markers,

• Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; Increasing protein production;

• Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load,

• Recombinant protein production in yeast: Saccharomyces cerevisiae expression systems

• Mammalian cell expression vectors: Selectable markers;

Fusion Proteins• Proteins created through the joining of two or more genes

which originally coded for separate proteins. • Translation of this fusion gene results in a single

polypeptide with functional properties derived from each of the original proteins.

• Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics.

• Chimeric mutant proteins occur naturally when a large-scale mutation, typically a chromosomal translocation, creates a novel coding sequence containing parts of the coding sequences from two different genes.

• Naturally occurring fusion proteins are important in cancer, where they may function as oncoproteins.

• Recombinant fusion proteins are created by removing the stop codon from a cDNA sequence coding for the first protein and appending cDNA sequence of the second protein in frame through ligation or overlap extension PCR.

• That DNA sequence express as a single protein. The protein can be engineered to include the full sequence of both original proteins, or only a portion of either.

• If the two entities are proteins, often linker (or "spacer") peptides are also added which make it more likely that the proteins fold independently and behave as expected.

• In the fusion protein where the linkers enable protein purification, linkers in protein or peptide fusions are sometimes engineered with cleavage sites for proteases or chemical agents which enable the liberation of the two separate proteins.

• This technique is often used for identification and purification of proteins, by fusing a GST protein, FLAG peptide, or a hexa-His peptide ( 6x His-tag) which can be isolated using nickel or cobalt resins by affinity chromatography).

• Chimeric proteins can also be manufactured with toxins or antibodies attached to them in order to study disease development.

Glutathione S-transferase (GST) gene fusion system.

Here, GST is used to purify and detect proteins of interest.

In a GST gene fusion system, the GST sequence is incorporated into an expression vector alongside the gene sequence encoding the protein of interest.

Induction of protein expression from the vector's promoter results in expression of a fusion protein - the protein of interest fused to the GST protein.

This GST-fusion protein can then be purified from cells via its high affinity for glutathione.

The tag has the size of 220 amino acids (roughly 26 KDa), which, compared to other tags like the myc- or the FLAG-tag, is quite big.

It is fused to the N-terminus of a protein. However, many commercially-available sources of GST-tagged plasmids include a thrombin domain for cleavage of the GST tag during protein purification.

Purification by GST TagGST-fusion proteins can be produced in

Escherichia coli, as recombinant proteins. The GST part binds its substrate, glutathione.

Agarose beads can be crosslinked with glutathione, and glutathione-Agarose beads bind GST-proteins.

These beads are then washed, to remove contaminating bacterial proteins.

Adding free glutathione to beads that bind purified GST-proteins will release the GST-protein in solution.

Properties of fusion Proteins

• The functionality of fusion proteins is made possible by the fact that many protein functional domains are modular.

• In other words, the linear portion of a polypeptide which corresponds to a given domain, such as a tyrosine kinase domain, may be removed from the rest of the protein without destroying its intrinsic enzymatic capability.

FLAG-tag, or FLAG octapeptide, is a polypeptide protein tag that can be added to a protein using Recombinant DNA technology. It can be used for affinity chromatography, to separate recombinant, overexpressed protein from wild-type protein expressed by the host organism. It can also be used in the isolation of protein complexes with multiple subunits.

A Flag-tag can be used in many different assays that require recognition by an antibody. If there is no antibody against the studied protein, adding a FLAG-tag to this protein allows following the protein with an antibody against the Flag sequence. Examples are cellular localization studies by immunofluorescence or detection by SDS PAGE protein electrophoresis.

The peptide sequence of the FLAG-tag is as follows: N-DYKDDDDK-C. It can be used in conjunction with other affinity tags for example a polyhistidine tag (His-tag), HA-tag or myc-tag. It can be fused to the C-terminus and the N-terminus of a protein. Some commercially available antibodies recognize the epitope only in certain positions, e.g. exclusively N-terminal. However, other available antibodies are position-insensitive.

The Flag tag can readily be removed from proteins once they have been isolated, using the specific proteinase, enterokinase (enteropeptidase).

• Myc (cMyc) codes for a protein that binds to the DNA of other genes. When Myc is mutated, or overexpressed, the protein doesn't bind correctly, and often causes cancer.

• When a gene like Myc is altered to cause cancer, the cancerous version of the gene is called an oncogene. The healthy version of the gene that it is derived from is called a protooncogene.

• Myc gene encodes for a transcription factor that regulates expression of 15% of all genes through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs). Myc belongs to Myc family of transcription factors, which also includes N-Myc and L-Myc genes. Myc-family transcription factors contain the bHLH/LZ (basic Helix-Loop-Helix Leucine Zipper) domain.

• A mutated version of Myc is found in many cancers which causes Myc to be persistently expressed. This leads to the unregulated expression of many genes some of which are involved in cell proliferation and results in the formation of cancer. A common translocation which involves Myc is t(8:14) is involved in the development of a lymphoma. A recent study demontrated that temporary inhibition of Myc selectively kills mouse lung cancer cells, making it a potential cancer drug target

Maltose Binding Protein (MBP) is a part of the maltose/maltodextrin system of Escherichia coli, which is responsible for the uptake and efficient catabolism of maltodextrins. It is a complex regulatory and transport system involving many proteins and protein complexes.

It is used in purification of recombinant proteins. A fusion protein of MBP with the protein of interest (say protein X) can be created. The fusion protein binds to amylose columns while all other proteins flow through. The MBP-protein X fusion can be purified by eluting the column with maltose. Once the fusion protein is obtained in purified form the protein of interest (X) can be cleaved from MBP with a specific protease. Protein X can be separated from MBP by passing through the amylose column again.

The gag-onc fusion protein is a protein formed from a group-specific antigen ('gag') and that of an oncovirus ('onc'), such as C-jun.

bcr-abl fusion protein is an oncogenic fusion protein, and is considered to be the primary oncogenic driver of chronic myelogenous leukemia.

Tpr-met fusion protein is an oncogene fusion protein consisting of TPR and MET

Example of Fusion Protein

• Secretion signal peptide

• Marker peptide for binding to antibody

• Marker peptide cleaved by bovine intestinal enterokinase

Factor Xa Protease

Blood coagulation factor protease Xa cleaves only at a specific amino acid sequence

Gly Gly Ser Ile Glu Gly Arg

Engineer Xa site between two polypeptides which make up fusion protein

Fusion Protein Cloning Vector• ompF promoter,

initiation of translation and secretion signal

• Cloned segment in frame with secretion peptide and which puts lacZ in frame makes fusion protein and ß-galactosidase activity

Fusion Proteins as Tools of Protein Purification

• Fusion proteins can simplify purification of recombinant proteins

• Can even do this and add stability on occasion

• Fused peptide commonly binds to a specific ligand during purification process

Some Fusion Systems Used to Facilitate the Purification of Foreign Proteins Produced in E.

Coli

Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load,

Increasing Protein Secretion

• Proteins in periplasm easier to purify

• Protein often more stable in periplasm

• Secretion to periplasm require signal sequence

• Gram-negative cells secrete very little

• Use either Gram-positive, eukaryotic or engineered Gram-negative cells

Protein Secretion to Periplasm

• Signal peptide binds to membrane receptors and the growing polypeptide is passed through

Engineering For Secretion

• Maltose Binding Protein secretion signal insufficient so entire gene used as fusion component

• Xa cleavage site added

• Cloned Gene (IL-2) released by cleavage

Increasing Secretion Efficiency

• Avoid cells which secrete endogenous proteases

• High level translation can actually inhibit secretion– Also may give incompletely processed

products– Using systems which over express

secretory genes can increase secretion and processing efficiency

Translation expression vectors In addition to explaining the features of ideal cloning vectors, translation

expression vectors used in mammalian cells for stable or transient expression of cDNAs in mammalian cells have

Strong CMV promoter for high-level, constitutive expressionHA epitope tag for convenient detection of expression productsSfi I sites for convenient, directional cloning of full-length cDNAs

Sfi I is a rare cutter enzyme which recognizes the sequence GGCCNNNNNGGCC. The variable core sequence allows the design of multiple Sfi I sites with incompatible overhangs within the same vector.

Dualsystems vectors feature two Sfi I sites with the sequences GGCCATTACGGCC and GGCCGCCTCGGCC, allowing directional cloning of cDNAs. Sfi I sequences are extremely rare in eukaryotic genomes and therefore, the majority of eukaryotic cDNAs can subcloned full-length using Sfi I.

pHA-MEX is mammalian expression vector featuring a strong CMV promoter, an N-terminal HA epitope tag for detection of expressed proteins and a NeoR cassette for stable integration of the expression vector into the genome of the host cell. pHA-MEX is suited for both transient overexpression and long-term, stable expression.

pMEX-HA is mammalian expression vector featuring a strong CMV promoter, a C-terminal HA epitope tag for detection of expressed proteins and a NeoR cassette for stable integration of the expression vector into the genome of the host cell. pMEX-HA is suited for both transient overexpression and long-term, stable expression.

Two gene expression vectors

Two Plasmid vectors are transfected simultaniously

E.g. pBICEP-CMV™ Expression Vector Amp resistant, 5.3 kb from Sigma Aldrich

For transient, cytoplasmic expression of an N-terminal FLAG® fusion and a second gene of interest or selection marker from bicistronic mRNA. Two genes can be cloned into MCS1 and MCS2 for transcription of a single message from the CMV promoter. This vector is useful for protein-protein interaction studies, multi-subunit proteins, and cloning a selection marker of choice.

Metabolic load

• The expression of a foreign protein(s) in a recombinant host cell or organism often utilizes a significant amount of the host cell's resources, removing those resources away from host cell metabolism and placing a metabolic load (metabolic drain, metabolic burden) on the host.

• As a consequence of the imposed metabolic load, the biochemistry and physiology of the host may be dramatically altered.

Metabolic load

• The numerous physiological changes that may occur often lowers the amount of the target foreign protein that is produced and eventually recovered from the recombinant organism.

DNA integration into bacterial genome

• Bacteria reproduce by the process of binary fission. In this process, the chromosome in the mother cell is replicated and a copy is allocated to each of the daughter cells.

• As a result, the two daughter cells are genetically identical. If the daughter cells are always identical to the mother, how are different strains of the same bacterial species created?

• The answer lies in certain events that change the bacterial chromosome and then these changes are passed on to future generations by binary fission.

Methods of DNA integration in bacterial genome are

Recombination

Genetic recombination refers to the exchange between two DNA molecules.

It results in new combinations of genes on the chromosome.

You are probably most familiar with the recombination event known as crossing over.

In crossing over, two homologous chromosomes (chromosomes that contain the same sequence of genes but can have different alleles) break at corresponding points, switch fragments and rejoin.

RecombinationThe result is two recombinant chromosomes.

In bacteria, crossing over involves a chromosome segment entering the cell and aligning with its homologous segment on the bacterial chromosome.

The two break at corresponding point, switch fragments and rejoin.

The result, as before, is two recombinant chromosomes and the bacteria can be called a recombinant cell.

The recombinant pieces left outside the chromosome will eventually be degraded or lost in cell division.

But one question still remains...how did the chromosome segment get in to the cell?

The answer is Genetic Transfer!

Genetic Transfer

Genetic transfer is the mechanism by which DNA is transferred from a donar to a recipient.

Once donar DNA is inside the recipient, crossing over can occur.

The result is a recombinant cell that has a genome different from either the donar or the recipient.

In bacteria genetic transfer can happen three ways:

Transformation

Transduction

Conjugation

Remember that a recombination event must occur after transfer in order that the change in the genome be heritable(passed on to the next generation).

Transformation

After death or cell lyses, some bacteria release their DNA into the environment.

Other bacteria, generally of the same species, can come into contact with these fragments, take them up and incorporate them into their DNA by recombination.

This method of transfer is the process of transformation.

Any DNA that is not integrated into he chromosome will be degraded.

The genetically transformed cell is called a recombinant cell because it has a different genetic makeup than the donar and the recipient.

All of the descendants of the recombinant cell will be identical to it.

In this way, recombination can give rise to genetic diversity in the population.

Griffith's Experiment

The transformation process was first demonstrated in 1928 by Frederick Griffith.

Griffith experimented on Streptococcus pneumoniae, a bacteria that causes pneumonia in mammals.

When he examined colonies of the bacteria on petri plates, he could tell that there were two different strains.

The colonies of one strain appeared smooth.

Later analysis revealed that this strain has a polysaccharide capsule and is virulent, that it, it causes pneumonia.

The colonies of the other strain appeared rough.

This strain has no capsules and is avirulent.

When Griffith injected living encapsulated cells into a mouse, the mouse died of pneumonia and the colonies of encapsulated cells were isolated from the blood of the mouse.

When living nonencapsulated cells were injected into a mouse, the mouse remained healthy and the colonies of nonencapsulated cells were isolated from the blood of the mouse.

Griffith then heat killed the encapsulated cells and injected them into a mouse.

The mouse remained healthy and no colonies were isolated.

The encapsulated cells lost the ability to cause the disease.

However, a combination of heat-killed encapsulated cells and living nonencapsulated cells did cause pneumonia and colonies of living encapsulated cells were isolated from the mouse.

How can a combination of these two strains cause pneumonia when either strand alone does not cause the disease?

If you guessed the process of transformation you are right!

The living nonencapsulated cells came into contact with DNA fragments of the dead capsulated cells.

The genes that code for thr capsule entered some of the living cells and a crossing over event occurred.

The recombinant cell now has the ability to form a capsule and cause pneumonia.

All of the recombinant's offspring have the same ability.

That is why the mouse developed pneumonia and died.

Transposons

Transposons (Transposable Genetic Elements) are pieces of DNA that can move from one location on the chromosome another, from plasmid to chromosome or vice versa or from one plasmid to another.

The simplest transposon is an insertion sequence.

An insertion sequence contains only one gene that codes frotransposase, the enzyme that catalyzes transposition.

The transposase gene is flanked by two DNA sequences called inverted repeats because that two regions are upside-down and backward to each other.

Transposase binds to these regions and cuts DNA to remove the gene.

The transposon can enter a number of locations.

When it invades a gene it usually inactivates the gene by interrupting the coding sequence and the protein that the gene codes for.

Luckil, transposition occurs rarely and is comparable to spontaneous mutation rates in bacteria.

Complex transposons consist of one or more genes between two insertion sequences.

The gene, coding for antibiotic resistance, for example, is carried along with the transposon as it inserts elsewhere.

It could insert in a plasmid and be passed on to other bacteria by conjugation.

Plasmids

Plasmids are genetic elements that can also provides a mechanism for genetic change.

Plasmids, as we discussed previously, are small, circular pieces of DNA that exist and replicate separately from the bacterial chromosome.

We have already seen the importance of the F plasmid for conjugation, but other plasmids of equal importance can also be found in bacteria.

One such plasmid is the R plasmid.

Resistance or R plasmids carry genes that confer resistance to certain antibiotics. A R plasmid usually has two types of genes:

R-determinant: resistance genes that code for enzymes that inactivate certain drugs

RTF (Resistance Transfer Factor): genes for plasmid replication and conjugation.

Without resistance genes for a particular antibiotic, a bacterium is sensitive to that antibiotic and probably destroyed by it.

But the presence of resistance genes, on the other hand, allows for their transcription and translation into enzymes that make the drug inactive.

Resistance is a serious problem. The widespread use of antibiotics in medicine and agriculture has lead to an increasingnumber of resistant strain pathogens.

These bacteria survive in the presence of the antibiotic and pass the resistance genes on to future generations.

R plasmids can also be transferred by conjugation from one bacterial cell to another, further increasing numbers in the resistant population.