Transformation/transfection

DNA of Prokaryotic CellsUNIT 3 Chapter 5: The Structure and Function of DNA Section 5.1

This is a partial map of the E. coli genome. There are actually thousands of genes in the genome, very few of which are non-essential.

The majority of prokaryotic genomes are composed of regions that contain either genes or regulatory sequences. Regulatory sequences are sections of DNA sequences that determine when certain genes and associated cell functions are activated.

Most prokaryotes are haploid, meaning they only have one set of chromosomes and therefore carry only one copy of each gene. Their genomes carry very little non-essential DNA.

RECOMBINANT DNA TECHNOLOGY : cuts

Treatment of a plasmid with an uniqueEcoR1 site. This restriction enzyme will open the plasmid and make it amenable for manipulation.

Principle of cloning a foreign DNA fragment

Transformation - Mechanisms

• Bacteria– transformation refers to a genetic change brought

about by picking up naked strands of DNA and expressing it, and competence refers to the state of being able to take up DNA.

Artificially competent cells of standard bacterial strains may also be purchased frozen, ready to use.Common Strain of E. coli - DH5α (alpha)

• Bacteria - Artificial competence - Temperature– Chilling cells in the presence of divalent cations such as

Ca2+ (in CaCl2) prepares the cell walls to become permeable to plasmid DNA.

– Cells are incubated with the DNA and then briefly heat shocked (42oC for 30-120 seconds), which causes the DNA to enter the cell.

• Divalent cations formed coordination complexes with the negatively charged DNA and LPS. However, DNA is a large molecule and it was not known how the DNA crossed the cell membrane to enter the cytosol.

• Recent work shows that the heat-shock step strongly depolarizes the cell membrane of CaCL2-treated cells

• The decrease of membrane potential may lower the negativity of the cell’s inside potential and allow the movement of negatively charged DNA into the cell’s interior.

• A subsequent cold-shock raises the membrane potential to its original value.

Transformation Procedure

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Results

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Transformation - Mechanisms

• Bacteria - Artificial competence – Electroporation– Electroporation is another way to make holes in cells,

by briefly shocking them with an electric field of 100-200V/cm.

– Now plasmid DNA can enter the cell through these holes.

– Natural membrane-repair mechanisms will close these holes afterwards.

Bacteria - Artificial competence – Lipofection Lipofection (or liposome transfection) is a technique used to inject genetic

material into a cell by means of liposomes which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.

The vescicle fuses with the cell membrane (similar to how two oil spots at the top of a broth will fuse) and the contents of the vesicle & the cell are combined.

Adenoviral vectors are useful for gene transfer due to a number of key features: 1) they rapidly infect a broad range of human cells and can achieve high

levels of gene transfer compared to other available vectors;

2) adenoviral vectors can accommodate relatively large segments of DNA (up to 7.5kb) and transduce these transgenes in nonproliferating cells;

3) adenoviral vectors are relatively easy to manipulate using recombinant DNA techniques (Vorburger and Hunt, 2002). Other vectors of interest include adeno-associated virus, herpes simplex virus, retroviruses and lentiviruses, a subset of the retrovirus family. Lentiviruses (e.g., HIV-1) are of particular interest because they are well studied, can infect quiescent cells, and can integrate into the host cell genome to allow stable, long-term transgene expression (Anson, 2004).

Formulation of Biotech Products, IncludingBiopharmaceutical Considerations

Such as

• Sterility• Solubility

• Choice of the delivery systems.• The route of administration.• Possibilities for target site-specific delivery of

proteins.

MICROBIOLOGICAL CONSIDERATIONS

• Sterility

• Protein pharmaceuticals have to be assembled under aseptic conditions

• Equipment's are treated separately and autoclaved, or sterilized by dry heat (>160 C), chemical treatment or gamma radiation to minimize the bioburden

• Filtration techniques are used for removal of microbacterial contaminants.

• The final “sterilizing” step before filling the vials is filtration through 0.2 or 0.22mm membrane filters.

Preservatives

• Antimicrobial agents are the mercury-containing phenylmercuric nitrate and thimerosal and p-hydroxybenzoic acids, phenol, benzyl alcohol and chlor-obutanol.

• The use of mercury containing pre-servatives is presently under discussion (FDA, 2006)

Pyrogen Removal

• Bacterial pyrogens are mainly endotoxins lipopolysaccharides from gram-negative bacteria.

Basic conserved structure of endotoxins : Lipid part, their high negative electrical charge

• Amphipathic in nature: Tendency to aggregate in water and their tendency to adsorb to surfaces.

• Stable under standard autoclaving conditions, but break down when heated in the dry state 160 C for prolonged periods(e.g., 30 minutes dry heat at 250C).

• Proteins may have a tendency to aggregate and precipitate.• Solutions : 1- proper pH and ionic strength conditions2- Addition of amino acids such as lysine or arginine

Insulin in solution is in an equilibrium state between monomeric, dimeric tetrameric, and hexameric forms.

The relative abundance of the different aggregation states depends on the pH, insulin concentration, ionic strength, and specific components (e.g., Zn and phenol)

• Solubility Enhancers

Anti-adhesion agents can also act as anti-aggregation agents

Albumin has a strong tendency to adsorb to surfaces and is therefore added in relatively high concentrations (e.g., 1%) to protein formulations as an anti-adhesion agent.

Insulin can form fibrillar precipitates (long rod-shaped structures with diameters in the 0.1mm range). Solutions:-Low concentrations of phospholipids and surfactants.-proper pH can also help to prevent this .

Surfactants can also prevent adhesion to interfaces and precipitation. These molecules readily adsorb to hydrophobic interfaces with their own hydrophobic groups and render this interface hydrophilic by exposing their hydrophilic groups to the aqueous phase.

• What are surfactants

• Common surfactants :

• Poloxamer 407, Poloxamer 188, polysorbate 80, polysorbate 20, octoxynol-9, polyoxyethylene-(23) lauryl alcohol, polyxyethylene-(20) oleyl alcohol, sodium lauryl sulphate.

Antioxidants

• Several amino acids like methionine, cysteine, tryptophan, tyrosine, and histidine are readily oxidized then all the protein is liable to oxidative degradation.

• Solutions • Replacement of oxygen by inert gases in the vials .• Anti-oxidants such as ascorbic acid or acetylcysteine . But Ascorbic acid act

as an oxidant in the presence of a number of heavy metals.

Shelf Life of Protein-Based Pharmaceuticals

• Proteins can be stored (i) As an aqueous solution. (ii) In freeze-dried form.(iii) in dried form in a compacted state (tablet).

Stability of protein solutions strongly depends on factors such as pH, ionic strength, temperature, and the presence of stabilizers.

Freeze Drying of Proteins• Water promotes chemical and physical degradation for proteins therefore

proteins in solution often has limited shelf life (>2 years).

• During freeze-drying water is removed through sublimation and not by evaporation.

• Three stages in the freeze-drying process: (i) Freezing step.(ii) The primary drying step.(iii) The secondary drying step.

In freezing and during the crystallization step ( -15c), protein concentration will increase

Minus forty degrees Celsius is a typical freezing temperature before sublimation is initiated through pressure reduction.

Freezing

This will concentrate the proteins and separate the water as crystals

Fast cooling is better, small crystals easier for sublimation

Primary Drying

• By lowering pressure• Water vapour condensed on condenser• Gradually increase the temperature to reach 2-5 C• This will remove the unbound water

• Secondary Drying • Increase temperature gradually to reach 20• This will leave 1% of the water content only

• Lyoprotectants are stabilizers used to prevent denaturation of proteins during freeze-drying and subsequent storage.