Protein engineering

Question 1

protein engineering uses

Answer 1

pharmaceuticals, agriculture, bioenergy, research, environmental, metabolic pathways

Question 2

problems with proteins

Answer 2

readily denatured, low activity, inappropriate activity

Question 3

rational protein design

Answer 3

target using structure and sequence knowledge -> mutations -> screen sequence -> structure -> function/behaviour
information intensive

Question 4

directed evolution

Answer 4

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

Question 5

subtilisin

Answer 5

laundry detergent = serine peptidase
alkali resistant
catalytic triad, oxyanion hole, specificity pocket, main chain substrate binding

Question 6

greater activity

Answer 6

broader substrate specificity, change binding pocket/tertiary structure

Question 7

changing sequence

Answer 7

order primers -> change sequence
plasmid with selected gene
PCR amplification -> select for change
sequencing to confirm

Question 8

increase stability

Answer 8

stop oxyanion hole functioning - oxidation

loops - add proline to decrease cleavage

Question 9

good target for engineering

Answer 9

easy kinetics/michaelis menton
mechanism well understood
crystallised enzyme
high expressed and secreted

Question 10

H lanuginosa lipase

Answer 10

triacylglycerol -> FFA
household detergents
low expression (not secreted)
interface catalysis mechanism (rolls to open to lipid soluble)

Question 11

engineering aims

Answer 11

improve expression, decreases specificity, increase activity, decrease protease sensitivity, stability, compatibility

Question 12

subtilisin E -> methyl formamide

Answer 12

both serine proteases (directed evolution)

Question 13

Error prone PCR

Answer 13

dGTP:dATP ratio -> more GTP nucleotide
introduces diversity
clone into bacterial plasmid
screen genotype plates -> phenotype plates (assay)
amplify lead/parent molecule

Question 14

DNA shuffling

Answer 14

break and reassemble different parent strands

Question 15

subtilisin evolution

Answer 15

10 mutations in loops surrounding catalytic site surface

more efficient in DMF

Question 16

types of changes

Answer 16

beneficial, deleterious, neutral

deconvolution

Question 17

sitagliptin

Answer 17

dipeptidyl peptidase-4 (DPP-4) inhibitor
decrease blood sugar in type 2 diabetes
oral + no hypoglycaemia

Question 18

DPP-4

Answer 18

chops GLP-1 incretin to stimulate insulin secretion

Question 19

transaminase engineering

Answer 19

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

Question 20

check product

Answer 20

oligerimisation SEC or mass spec

model structure

Question 21

antibodies

Answer 21

Fv - variable region
Fc - constant region
Fab - one constant one heavy
3 x heavy and light chains = binding

Question 22

phage display

Answer 22

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

Question 23

antibody phage display

Answer 23

linker between heavy and light chains (sFv - single chain variable constant) made from gly and ser
select for binding to ligand

Question 24

technological advancements for selection of antibodies

Answer 24

monovalent display - only one copy on surface (shows binding better)
effective library size - bigger = tight binding (don’t need mature proteins)

Question 25

adalimumab/humira

Answer 25

treat inflammatory diseases
blocks binding to TNF (tumor necrosis factor) - activates inflammation, validated target
extracellular = good for antibodies

Question 26

mouse antibodies

Answer 26

good for acute not chronic

human antibodies develop against

Question 27

human mAb templated by mouse mAb

Answer 27

heavy chain mice fuse with human light chain -> screen for best
good light chain with human heavy chain -> rescreen
= human heavy and light chain

Question 28

CDR

Answer 28

Complementarity-determining regions

core of binding domain, variable = recognition

Question 29

infliximab

Answer 29

chimeric/humanised mouse antibody for inflammatory disease

not as effective in blocking site - wrong placement