Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

B3.0 > Protein Engineering > Flashcards

Protein Engineering Flashcards

1
Q

Structure

A
  1. Intro
  2. Structure-function relationships
    3.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

engineering

A
  • teleological
  • specific
  • deliberate
  • optimised
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

protein engineering

A

modifying a protein sequence for better suitability

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

evolution

A

molecular tinkering

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

KM

A

ES stability

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

KCAT

A

rate of S->P conversion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

KCAT/KM

A
  • catalytic efficiency
  • highly biologically relevant
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

VMAX

A

maximum KCAT

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How do you use protein engineering to understand structure-function relationships

A

1) insert gene of interest into plasmid cloning vector
2) insert primer w/ desired mutated sequence; strand separation and synthesis
3) digest DNA
4) transform bacteria
5) generate daughter cells (contain mutant DNA)
7) centrifuge soluble protein
8) purify
9) Western blot (SDS-Page)
10) functional assay

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

SARS CoV-2 3CL protease clinical variant P108S

A
  • decreased clinical severity in Japanese cohort
  • kinetic and biochemical assays showed weakened substrate binding (Kmx2)
  • engineering recombinant protein allows correlation of different structural properties with clinical outcome
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

proline changes

A

significant for both primary and secondary structure

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

3CL protease

A
  • necessary for viral replication
  • therapeutic target
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

PCOs

A
  • H164D: non-functional (decreased iron interaction)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

rational mutagenesis

A

using detailed protein structural and functional knowledge to make educated engineering decisions

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

RUBISCO

A
  • 0.7Gt
  • multi-domain recombinant productjon
  • requires chaperones
  • diverse amoungst photosynthetic organisms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

describe the multi-domain recombinant production of RUBISCO

A

different subunits nuclear (RbcS) and chloroplastic (RbcL)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Describe the photosynthetic hierarchy

A

red algae > plants > green algae > cyanobacteria

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

“red lineage RUBISCO”

A
  • unknown chaperones
  • R. sphaeroides: transformable
  • G. monolis: high efficiency
  • chimaeric recombinant protein has high selectivity and KCAT
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Rhodobacter sphaeroides

A

proteobacterium

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Griffithsia monolis

A
  • resolved crystal structure
  • loop 6 encloses AS
  • highly conserved
  • A331, V334
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Rational mutagenesis of RUBISCO in vivo

A
  • proof-of-principle
  • in tobacco
  • increased photosynthetic rate (micromole /s/m2)
  • increased plant height (cm)
  • decreased growth (due to inefficient expression)
  • apply to endogenous RUBISCOs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

4pco

A
  • increased anaerobic gene expression
  • not ideal due to multifunctional PCOs
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

4pco + modified PCO4

A
  • increased anaerobic gene expression
  • mixed fertility
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

rational mutagenesis to combat inherited disease

A
  • e.g. hypertrophic cardiomyopathy
  • R403Q in beta-myosin heavy chain (an important ESP)
  • correct using ABE + gRNA + cardiac promotor
  • decreased hypertrophy phenotype
  • increased ejection fraction
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
hypertrophic cardiomyopathy
- most common cause of sudden cardiac death in adolescents and young adults - commonly found inherited mutations in heart sarcomere genes
26
ABE
- adenine base editor - uses AAV vector
27
AAV
adeno-associated virus
28
Next steps for rational mutagenesis to target hypertrophic cardiomyopathy
- animal models and clinical trials
29
"ABE might be a
one-time therapy to permanently correct HCM-causing variants, and prevent disease onset"
30
Describe base editing in CRISPR
1) Cas makes a nick 2) base editing enzymes
31
base editing enzymes
cytidine/adenosine deaminase
32
Describe prime editing in CRISPR
- Cas9 makes a nick - prime editing gRNA + primer BD conjugated to RT; binding - Fen1: flap repair
33
General challenges for rational mutagenesis to combat inherited disease
- correlating in vitro with in vivo - missing technologies for rapid screening - off-target effects - safe, effective delivery methods - ethical concerns
34
Which inherited diseases are currently being tackled using rational mutagenesis?
- Duchenne muscular dystrophy - cystic fibrosis - sickle cell anaemia
35
Deep mutational scanning
- aka: site-saturation mutagenesis - replacing every aa in a protein w/ every alternative - easiest in prokaryotes - identifies "unpredictable" desirable variants for further testing
36
Deep mutational scanning of RUBISCO
- mutated all R. rubrum residues - Delta-rpi E. coli model: screen function - change CO2 availability - screen for higher affinity - reveals hotspots for future engineering
37
Directed evolution
- using strategies to randomly introduce mutations into exonic sequences, functionally screening outcomes, then iterating for optimisation - aka "automated mutagenesis" - easiest in prokaryotes
38
directed evolution + biocatalysis
1) random mutations introduce in gene of interest via error-prone PCR 2) genes inserted into bacteria 3) enzymes tested and efficiency selected for
39
directed evolution can be used for
- biocatalysts - designer Abs
40
designer Abs
- requires Ab library - rheumatoid arthritis
41
directed evolution and Ab selection
1) genetic info for Ab binding site instead into phage dna 2) selection for Abs w/ strong attachments 3) random mutations introduced to bound Abs 4) increased affinity and specificity
42
phages
- efficient gene delivery and self amplification - high mutation rate
43
Coupling biocatalysts and ab selection
- combining desirable function in the BOI with successful phenotype allows continuous evolution; fast!
44
In the lagoon
- constant inflow of fresh bacterial cells w/ mutagenesis + accessory plasmids - phage infection results in mutagenesis and gene expression - if BOI is functional, it interacts with RNAP; allows next cycle of infection alongside increase in functional properties - if BOI is non-functional, it leaves the lagoon
45
Directed evolution and carbonic anhydrase
- carbon capture and sequestration - optimise temperature sensitivity - 9 rounds formed a temperature sensitive + alkaline-tolerant variant - allows solvent reuse
46
Directed evolution and PETase
- chemotrypsin-like - bacteria evolved on okastuc recycling facility - PET is C source
47
HotPETase
hydrolyses semi-crystalline PET
48
Describe the directed evolution of HotPETase
i) array enzyme libraries in 96 deep-well plates ii) carry out reactions iii) incubate PET disc w/ enzyme to release soluble products iv) quench and precipitate v) quantify soluble degradation product (MHET, TPA by UPLC)
49
OrthoRep
- use yeast to develop bacterial O2-tolerant THI4, for use in plants
50
THI4
- enzyme - forms thiamine precursor - most efficient in anaerobic bacteria
51
How did OrthoRep work?
- cells selected for in thiamine-free media - THI4-V12A is able to grow
52
Synthetic biology - generally
- design and construction of new biological parts/ modifying existing biological systems - protein engineering is a subset of this
53
Synthetic biology - here
- adding unnatural amino acids to existing proteins - can be incorporated in vivo using amber stop codons/ engineered tRNAs
54
de novo protein design
- using in silico strategies to generate a protein sequence that is complementary to a known POI - validate in vitro and in vivo
55
RFdiffusion
- generates sequence for protein binding - produces recombinant protein w/ high affinity interactions - actual structures recapitulate predicted
56
RFdiffusion + AlphaFold2
- generate and filter sequence for anti-venoms
57
Antivenoms in vitro
- strong interaction - structure as expected
58
Antivenoms in vivo
- cells and mice - protective effect
59
AlphaFold 1
- "fed" physical + evolutionary info - low resolution - worse than experimental data
60
AlphaFold 2
- "learns" physical and evolutionary info - high resolution - as good as experimental data
61
AlphaFold Multimer
- uses a "linker" technique to predict protein interactions - also RosETTA fold
62
AlphaFold
- combines simulation of physical interactions with evolutionary modelling - pairs of interacting molecules are conserved if important
63
AlphaFold 3
- models individual atoms (not aas) - can predict protein complicies - not so good for proteins wit unknown evolutionary relatives
64
AI-supported protein engineering
- AlphaFold Multimer - ePip1 - in vitro: insensitive - in vivo (N. benthamiana): increased resistance
65
Pip1
- ETI - apoplastic immune protease inhibited by P. infestans EpiC2b

B3.0 (44 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions