GEN #14 Recombinant Protein Methods
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Banting & Best isolated insulin enriched fluid from _____ of dogs whose pancreatic duct had been _______.
Insulin Protein is a ________ primary gene product (INS gene).
Insulin Protein is processed by ________ and _________to remove the __________ portion.
- prohormone convertase-1 & -2
The final insulin protein has two chains _____ & _______ linked by ________bridge
Insulin protein is highly conserved with _____ difference for porcine insulin and ______ difference for bovine
In addition to a disulfide bridge the A & B chains are also linked by a _____ _______
The expression vector to use will depend on
- the size of the cDNA to be inserted
- the host cell into which the vector will be introduced
- the need for production of a fusion protein
Bacterial cells preferred for recombinant proteins because
- E. Coli genetics well understood
- Large amounts of protein expressed
- Fermentation technology advanced
Yeast or COS cells are
- Simplest of eukaryotic cells
- Can grow in large bioreactors
COS cells have to grow on
The two major problems with E. Coli production of proteins
- Inclusion bodies
- Solubility of proteins may be low
Low solubility of proteins is why the protein may need to be ______ to a _______ __________ that can be removed later in the processing.
The recombinant protein needs to be purified
from all ________ ____________
The recombinant protein purity should be
The recombinant protein should be kept in its
The ______ of the recombinant protein should be maintained
Protein is released form the cells by physically or chemically _________ _______ the cells
Stabilize the proteins and protect from ________
__________ such as _________ _________ ___________ or ________ _________ may be needed to keep the protein soluble during purification
- sodium dodecyl sulfate
- sodium desoxycholate
_________ selectively removes large molecules from a mixture
Precipitation may lead to ________ ___________
Commonly used separating agents include ________ ______ and __________ ________
- ammonium sulfate
- sodium chloride
_______ is done on a molecular sieve sized basis
____________ is the technique of choice for separating proteins
Chromatography can separate molecules based on
- molecular weight [size exclusion]
- ionic charge [ion exchange]
- hydrophobicity interaction
- specific functionality (affinity chromatography)
The AA sequence should not be _________
___________ is used to ensure the protein is in the correct 3D conformation.
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