16 - Protein Engineering Flashcards
protein engineering
- used to make natural tools more useful tools
- rational methods
- non-rational methods
subtilisin
- protease that removes protein-based stains by chopping up the protein
protein design
- can be used to improve the stability of an enzyme
- changes an amino acid close to a catalytic region
GFP revolution
allows biological processes to be highlighted
GFP mutation
- highly mutated through rational engineering
- changing bases to His, Trp, Phe, or Tyr will change the luminescence colour of the protein
- mutating Ala to a larger, charged protein will repel dimers and produce a monomer
rational protein engineering
using science based knowledge to alter proteins to be more useful
non-rational protein engineering
- libraries of variants mutated and the successful variants are replicated
- empirical strategy where the solution is not known before-hand
- directed evolution
human therapeutic antibodies
- therapeutic antibodies must be human antibodies that bind to human protein
- human immune system is unable to make antibodies against human proteins
- microbial surface display
- transgenic mouse
- human memory B-cell immortalization
- humanization
microbial surface display
- libraries of antibodies displayed on the surface are examined
- links function with gene, therefore human antibodies can be created from it
transgenic mouse
- mice immune system consists of human antibodies after human gene injection
- human antibodies are made from the mice antibodies
human memory B-cell immortalization
isolate memory cells of an infected patient that survived, and make antibodies for another
humanization
mice binding/recognition site are inserted into human genes
avastin
- antibody used against tumors
- binds to vascular endothelial growth factor (VEGF) to prevent it from binding to the receptor
vascular endothelial growth factor (VEGF)
- released by tumors
- stimulates angiogenesis
angiogenesis
formation of new blood cells
phage display
displaying foreign peptide on the surface of a filamentous phage
filamentous phage
tubes with DNA that encode protein for structure
phage display mechanism
- insert genome into phage
- transform prophage vectors to E. coli
- phages will express different antibodies
prophage
virus found in its DNA form, therefore it’s not infectious yet
in vitro affinity selections
- phage display done 3-5 times on successful variants
- repeated for amplification
- DNA sequenced is used to find the actual sequence of the amplified structure
Humira
- drug developed from phage display
- # 1 best selling drug
- binds to TNF-alpha to prevent it from binding with its receptor
- used to treat rheumatoid arthritis
rheumatoid arthritis
immune system attacks cartilage and breaks it down
infliximab
- synthesized through humanization
- works similarly to Humira (adalimumab)
- has more side effects
- decreased in sales
