Genetic Manipulation I Flashcards
(39 cards)
What are the major applications of plasmids?
- Expression of recombinant protein in different host cells, e.g. production of proteins
- Expression of mutant proteins in different host cells
- Manipulation of genetic expression
- Expression and study of the regulatory sequences (Promoter) of gene expression
Describe Application #1-Recombinant protein production
- Recombinant protein is a manipulated form of a protein encoded by a gene (recombinant DNA where the gene coding sequence is cloned in a system that supports expression of the gene and translation of messenger RNA).
- The recombinant protein can be in the wild type form or in a mutated form.
What is Posttranslational modification?
The covalent and generally enzymatic modification of proteins following protein biosynthesis.
Postranslational modification: Hydroxylation
Attaches a hydroxyl group (-OH) to a side chain of a protein
Postranslational modification: Methylation
Adds a methyl group, usually a lysine group or arginine residues
Postranslational modification: Lipidation
Attaches a lipid, such as a fatty acid, to a protein chain
Postranslational modification: Acetylation
Adds an acetyl group to an N-terminus of a protein or at lysine residues
Postranslational modification: Disulfide bonds
Covalently links the S atoms of two different cysteine residues
Postranslational modification: SUMOylation
Adds a small protein SUMO (small ubiquitin-like modifier) to a target protein
Postranslational modification: Ubiquitination
Adds ubiquitin to lysine residue of a target protein for degradation
Postranslational modification: Glycosylation
Attaches a sugar, usually to an N or O in an amino acid side chain
Postranslational modification: Phosphoyrlation
Adds a phosphate to serine, threonine or tyrosine
What are the pros of bacterial expression systems?
They require simple culture conditions (e.g. media and additives), which are scalable and incurs low cost
What are the cons of bacerial expression systems?
- Difficulty in expressing some mammalian proteins
- Protease contamination from host cells leading to degradation of expressed protein
- Endotoxin contamination
- Lack of post-translational modification
What are the pros of yeast expression systems?
- Well-defined, economic eukaryotic expression system
- Suitable for expression of secretory proteins as well as intracellular proteins
- High protein yield, lesser expression time, post- translational modifications and requires simple media
What are the cons of yeast expression systems
- Hyperglycosylation of proteins, unlike mammalian system, it offers both N and O linked oligosaccharides for glycosylated proteins
- Fermentation is necessary for high protein yield
What are the pros of insect cell expression systems?
- Most used technique to express mammalian proteins, which require post-translational modifications
- Most similar to mammalian expression systems
- Can be used both in adherent and suspension cultures
- Purification process is easy
- Baculoviruses are safe to work with compared to mammalian viruses
What are the cons of insect cell expression systems?
- Time consuming cloning procedure of desired gene in baculovirus vector
- Requiring very expensive media
- Glycosylation is different from that of mammalian system resulting in improper maintenance of epitopes on protein
What are the pros for mammalian expression systems?
- An ideal choice for the production of therapeutic proteins and vaccines
- Both plasmid or virus based vectors can be used
- Offering desired post-translational modifications and proper protein folding
What are the cons of mammalian expression systems?
- High cost of protein production, because of slow cell growth, expensive media and culture conditions (continuous CO2 supply, expensive transfection reagents)
- High protein yield can be obtained only by suspension cultures. e.g. Chinese Hamster Ovarian (CHO) cells and Human Embryonic Kidney (HEK 293) cells
What are the pros of cell free expression systems?
- Expressing proteins in a cell free environment using cell extract, DNA template, amino acids and cofactors
- A simple process, protein expression and purification can be done in a short period of time (1-2 days)
What are the cons of cell free expression systems?
- Degradation of protein by exogenous proteases from cell extracts
- The extracts are expensive
- Small yields
What are the considerations for recombinant protein rpdocution?
- Yield
- Costs
- Protein of interest, e.g. intracellular or extracellular protein, post-translational modification etc.
- Culture condition
- Contamination, e.g. endotoxin etc.
- Protein degradation
What is genetic manipulation?
- The process of inducing changes in gene expression and the expression of novel genes.
- Manipulation of gene expression allows us to study the impact of proteins on genes or other proteins.
