Expression and analysis and human G6PD

What is the function of human glucose-6-phosphate dehydrogenase (G6PD)?

Catalyzes the first and rate limiting step in the pentose phosphate pathway for the production of NADPH, which protects cells from oxidative damage.

How does G6PD deficiency affect red blood cells (RBCs)?

Reduced production of NADPH selectively affects RBCs as they rely solely on the pentose phosphate pathway for NADPH. Oxidative stress may damage the membrane proteins of RBCs, leading to hemolysis.

What are some triggers for acute hemolytic anemia in G6PD deficiency patients?

Injection of oxidant drugs, infections, and ingestion of fava beans.

How many mutations have been reported in G6PD deficiency?

Approximately 160 mutations have been reported.

What are the common G6PD variants in the Chinese population?

G6PD Canton (Arg459Leu), G6PD Kaiping (Arg463His), and G6PD Gaohe (His32Arg).

What is recombinant DNA?

Biologically active DNA formed by the in vitro joining of segments of DNA from different sources.

What is the purpose of recombinant DNA technology?

To clone genes and make recombinant proteins when introduced into host cells.

What is a restriction enzyme?

Restriction endonucleases that recognize and cut DNA at specific nucleotide sequences.

What are the two types of ends produced by restriction enzymes?

Cohesive (sticky) ends and blunt ends.

What is a cloning vector?

A replicating DNA molecule that carries a foreign DNA fragment to be introduced into a cell, such as plasmid DNA.

What are the properties of a cloning vector?

It replicates independently of the host cells, has unique restriction enzyme cleavage sites, and contains a reporter gene/selectable marker to determine which host cells contain the recombinant DNA.

What is an expression vector?

A cloning vector that includes sequences needed for the expression of the foreign DNA, such as a promoter, ribosome binding site, and transcription termination sequence.

What are the steps in the traditional cloning process?

Vector preparation, insert preparation, ligation, transformation, and colony screening.

What is plasmid subcloning?

The process of preparing insert DNA containing the coding sequence of G6PD and preparing vector DNA for cloning G6PD.

What is the purpose of the His-tag in the G6PD expression vector?

The His-tag is a tag made up of 6 consecutive histidine residues and allows for purification of the recombinant protein using affinity chromatography.

What are the steps involved in constructing a G6PD expression vector?

Digesting the parent vector and the vector DNA, ligating the G6PD DNA insert to the vector DNA, and ensuring proper expression using the vector-encoded T7 promoter.

What is the most commonly used bacteria for introducing recombinant expression plasmids?

E. coli.

How is DNA uptake achieved in bacteria during transformation?

DNA passes through channels formed at the adhesion zone and heat shock creates a thermal imbalance to help pump DNA via the adhesion zone.

What is transformation efficiency?

It is a measurement of the quality of competent cells and is defined as the number of colonies formed per μg of uncut plasmid DNA.

What are the methods used for identification of recombinant clones?

Restriction enzyme analysis, colony PCR, blue-white selection, and DNA sequencing.

How does blue-white selection work?

Bacteria that have taken up the plasmid grow in the presence of antibiotics, and only bacteria that carry the recombinant plasmid produce white colonies due to the presence of insert DNA containing lacZ.

Why is DNA sequencing used to confirm positive clones?

It ensures that the reading frame is correct and checks for any extra mutations that may have occurred during the cloning process.

What is the pET expression system?

It is an inducible system for high-level expression of recombinant proteins in bacteria.

How is the expression of the cloned gene controlled in the pET expression system?

It is controlled by the T7 promoter, specific to T7 RNA polymerase but not E. coli RNA polymerase.

What is the role of the T7 RNA polymerase in the pET expression system?

It is required for the expression of the cloned gene and is controlled by the operator site in the promoter.

What is the operator in the lac operon?

It is a sequence that regulates transcription by binding the repressor protein.

What is the function of the repressor protein in the lac operon?

It binds to the operator and blocks transcription of the operon.

What happens when lactose binds to the operator in the lac operon?

Transcription of the operon is enabled.

What is ITPG induction?

It is the induction of gene expression in the lac operon by removing the repressor protein from the operator.

What is auto-induction in protein expression?

It is the induction of gene expression when cells reach saturation, typically achieved by using a medium containing glucose and lactose.

What is catabolite repression?

It is the repression of lactose utilization enzymes by the presence of glucose, which reduces the concentration of cAMP and decreases the efficiency of operon transcription.

What is the purpose of cell extraction in protein purification?

It is the process of collecting and lysing cells to release the desired protein.

Name three methods of cell disruption in cell extraction.

Detergent or enzyme (lysozyme), sonication, and mechanical homogenization.

How can proteolysis be minimized during cell extraction?

By using protease inhibitors and performing the process at low temperatures.

What is selective precipitation in protein purification?

It is a method to separate proteins based on their solubility by adding ammonium sulfate or water-miscible organic solvents.

How does pH alteration affect protein precipitation in selective precipitation?

It can cause proteins to aggregate and precipitate at their isoelectric point.

What is the principle of chromatography in protein purification?

Components are separated based on their affinity to the stationary phase or their distribution between the stationary and mobile phases.

What is affinity chromatography?

It is a chromatographic method that relies on specific interactions between a protein and a ligand on the matrix.

Give an example of affinity chromatography and its elution condition.

Glutathione (ligand) and GST fusion protein (target protein) elute when exposed to reduced glutathione and low ionic strength.

How is a his-tagged protein purified using Ni-NTA agarose column chromatography?

The target protein binds to the column, and unbound material is eluted. The bound protein is then recovered by adding imidazole to favor elution.

What is ion-exchange chromatography?

It is a chromatographic method that separates proteins based on their electrostatic interaction with the matrix.

What is hydrophobic interaction chromatography?

It is a chromatographic method that separates proteins based on their surface hydrophobicity.

What is the principle of gel filtration chromatography?

It separates proteins based on their size, with larger proteins eluting first and smaller proteins being retarded differentially.

What is the purpose of dialysis or ultrafiltration in protein purification?

It is used to remove salts, exchange buffers, and concentrate proteins.

How is the concentration of a protein determined?

By measuring its absorbance at a specific wavelength using a spectrophotometer.

What is the purpose of SDS-PAGE in protein purification?

It is used to analyze the purity and molecular weight of the protein of interest.

How can protein purity be assessed?

By using techniques such as SDS-PAGE, Western blotting, or mass spectrometry.

What is the purpose of a biological activity assay?

To detect the protein.

What are the two types of biological activity assays?

Continuous assay and discontinuous assay.

How is a continuous assay measured?

By changes in absorbance, fluorescence, or pH.

How is a discontinuous assay measured?

Product is measured after the reaction is manually stopped or quenched at different times.

What is a coupled assay?

A method for detecting substrate-product pairs with spectroscopic properties.

How does a coupled assay work?

The product from the reaction catalyzed by the enzyme of interest is used as a substrate in a second enzymatic reaction, yielding a compound that can be directly detected.

Give an example of a coupled assay.

Measuring hexokinase activity by measuring the increase in NADPH.

How is G6PD activity measured by continuous assay?

By measuring the absorbance at 340 nm, which increases when NADPH accumulates.

What is enzyme activity measured in?

Units (U).

How is enzyme activity calculated?

(Change in absorbance at 340 nm per minute) * (Reaction volume) / (6.22 * Enzyme volume).

What is specific activity?

Activity per unit weight, indicating protein purity and quality.

How is specific activity calculated?

Enzyme activity (U) divided by protein weight (mg).

What is overall yield or recovery?

The total target protein or activity in a fraction divided by the total target protein or activity in the crude extract.

What is purification factor?

The specific activity of a fraction divided by the specific activity in the crude extract.

How is protein concentration determined using UV spectrometry?

By measuring the absorbance at 280 nm, which is influenced by the presence of tryptophan, tyrosine, and cystine in the protein.

What is the disadvantage of UV spectrometry for protein concentration determination?

It can only measure pure protein samples.

What is the Bradford method?

A dye-based method for protein concentration determination using Coomassie Brilliant Blue G-250.

How does the Bradford method work?

The dye binds to proteins, causing a color change that can be detected at 595 nm.

What is the disadvantage of the Bradford method?

It is affected by detergents in the sample.

What is the Lowry method?

A copper-based method for protein concentration determination.

How does the Lowry method work?

It involves the chelation of Cu2+ by peptide bonds and the reduction of Cu2+ to Cu+, which reacts with the Folin reagent to produce a blue product that can be measured at 750 nm.

What is the BCA method?

Another copper-based method for protein concentration determination using bicinchoninic acid.

How does the BCA method work?

Cu2+ in the BCA reagent forms complexes with peptide bonds, which are then reduced to Cu+ and react with BCA to form a purple complex that can be measured at 562 nm.

What is SDS-PAGE used for?

To check protein purity, estimate protein molecular mass, and for immunodetection of proteins.

What is the purpose of Western blotting?

To confirm the identity of a target protein and compare protein expression in different conditions.

How is protein separation visualized in a Western blot?

By staining the gel with Coomassie blue or colloidal gold.

What is the purpose of blocking unbound sites on a Western blot membrane?

To prevent non-specific binding of antibodies and reduce background signals.

What are primary antibodies?

Antibodies that bind to the target protein or a specific epitope on the protein.

What are secondary antibodies?

Antibodies that recognize and bind to the primary antibody, often conjugated to an enzyme or other detection molecule.

What is the difference between direct and indirect detection in Western blotting?

Direct detection involves labeling the primary antibody with an enzyme, while indirect detection uses an unlabeled primary antibody and relies on a secondary antibody for detection.

How is His-tagged G6PD detected in Western blotting?

Using a primary antibody against G6PD and a secondary antibody conjugated with alkaline phosphatase (AP), which produces a purple product when incubated with a substrate.

How is data visualized in Western blotting?

Using a CCD camera or by exposing the blot to X-ray film.