Flashcards in LECTURE 1 (PROTEIN PURE) Deck (69)
When we purify proteins we want to keep them in what kind of formation?
3D structure maintained
Proteins hydrophobic residues are localised mostly where?
Core, although there are hydrophobic patches
Purification of protein is all about ?
exploiting its individual properties
Until what point, can we look at the kinetics of an enzyme?
Purification percentage aim (moderate)
If a protein isn't pure it can cause ? (drugs)
unexpected side effects
examples of low cost protein production
industrial bulk enzyme
Antigen for antibody sequencing purification percentage ?
greater than 95%
If STRUCTURE and CHARACTERISATION is being explored of a protein, purification percentage must be ??
high (greater than 95)
Therapeutic protein use
greater than 99.9%
Aim of protein purification?
Aim: High yield retaining maximum activity
four steps of protein purification?
1) Preparation extraction and clarification
3) Intermediate purification
In order to purify a protein its properties must consist of which four things ?
- Readily available
- Wide range
Advantages of protein purification?
Formed in vivo
Disadvantages of protein purification- natural sources ?
Abundance and reproducibility
Plant sources often _______
low in abundance (proteins)
- oxidation occurring
- seeds in large quantities to get a large abundance
- Fruits undergo developmental changes (particular ripeness at particular times)
Recombinant sources for protein preparation?
Clone gene into a plasmid, overexpress
- Usually bacteria (e.coli)
- or transgenic animals
Yield of recombinant sources of protein production
yields may be 100-fold higher
When is recombinant sources of protein production most successful ?
In similar organism to which it originated from
how is over expression in pro's achieved?
Genes for protein of interest are under the control of a strong promotor
Advantages of Prokaryotic expression?
Rapid growth, simple nutrition
disadvantages of Prokaryotic expression?
No post- translational modifications
How to overcome insolubility of proteins?
Add detergent and refold the protein (not always successful)
Eukaryotic expression advantages?- YEAST -(S. cerevisiae)
- good growth rates
- Simple (cheap) media
- Genetics understood
Eukaryotic expression disadvantages?- YEAST (S. cerevisiae)
hyperglycosylation (adding too many glucose groups)
Pichia pastoris advantages vs disadvantages (eu expression)
higher cell density, grows on methanol, still misfolds
Insect cells driven expression by??
infect cells with baculovirus (which contains the gene of interest)>>>> protein production