Protein engineering Flashcards
protein engineering uses
pharmaceuticals, agriculture, bioenergy, research, environmental, metabolic pathways
problems with proteins
readily denatured, low activity, inappropriate activity
rational protein design
target using structure and sequence knowledge -> mutations -> screen sequence -> structure -> function/behaviour
information intensive
directed evolution
create different mutations and screen for best ones
intentional variation of protein sequences at a defined level of randomness
start with protein that has functional link (promiscuous region) -> expose to harsh environments
subtilisin
laundry detergent = serine peptidase
alkali resistant
catalytic triad, oxyanion hole, specificity pocket, main chain substrate binding
greater activity
broader substrate specificity, change binding pocket/tertiary structure
changing sequence
order primers -> change sequence
plasmid with selected gene
PCR amplification -> select for change
sequencing to confirm
increase stability
stop oxyanion hole functioning - oxidation
loops - add proline to decrease cleavage
good target for engineering
easy kinetics/michaelis menton
mechanism well understood
crystallised enzyme
high expressed and secreted
H lanuginosa lipase
triacylglycerol -> FFA
household detergents
low expression (not secreted)
interface catalysis mechanism (rolls to open to lipid soluble)
engineering aims
improve expression, decreases specificity, increase activity, decrease protease sensitivity, stability, compatibility
subtilisin E -> methyl formamide
both serine proteases (directed evolution)
Error prone PCR
dGTP:dATP ratio -> more GTP nucleotide introduces diversity clone into bacterial plasmid screen genotype plates -> phenotype plates (assay) amplify lead/parent molecule
DNA shuffling
break and reassemble different parent strands
subtilisin evolution
10 mutations in loops surrounding catalytic site surface
more efficient in DMF
types of changes
beneficial, deleterious, neutral
deconvolution
sitagliptin
dipeptidyl peptidase-4 (DPP-4) inhibitor
decrease blood sugar in type 2 diabetes
oral + no hypoglycaemia
DPP-4
chops GLP-1 incretin to stimulate insulin secretion
transaminase engineering
asymmetric hydrogenation
need to aminate prostiglipin ketone, R-selective, large pocket
directed evolution (site-saturating - one at a time)
move loop G136F and clip chains to open pocket
check product
oligerimisation SEC or mass spec
model structure
antibodies
Fv - variable region
Fc - constant region
Fab - one constant one heavy
3 x heavy and light chains = binding
phage display
DNA from phage library inserted -> present on phage surface
identify and amplify/repeat best binding proteins
use protein 3 (coat protein) to bind favourite protein
single polypeptides
antibody phage display
linker between heavy and light chains (sFv - single chain variable constant) made from gly and ser
select for binding to ligand
technological advancements for selection of antibodies
monovalent display - only one copy on surface (shows binding better)
effective library size - bigger = tight binding (don’t need mature proteins)
adalimumab/humira
treat inflammatory diseases
blocks binding to TNF (tumor necrosis factor) - activates inflammation, validated target
extracellular = good for antibodies
mouse antibodies
good for acute not chronic
human antibodies develop against
human mAb templated by mouse mAb
heavy chain mice fuse with human light chain -> screen for best
good light chain with human heavy chain -> rescreen
= human heavy and light chain
CDR
Complementarity-determining regions
core of binding domain, variable = recognition
infliximab
chimeric/humanised mouse antibody for inflammatory disease
not as effective in blocking site - wrong placement
