Protein Chemistry

PPT 10

Quiz ChatGPT conversation

🧬 Protein Selection Strategies — Full Summary

🧠 1. Overview of Selection Methods (p.1–2)

Core idea:

You want to find functional proteins (e.g., binders, enzymes) from huge libraries (up to ~10¹³ variants).

Main strategies:

Cell-based systems
- Complementation
- Yeast display
- Bacterial display
- Phage display
- Retroviral display
In vitro systems
- Ribosome display
- mRNA display
- CIS display
- Compartmentalisation

🔑 Key concept:

👉 Genotype–phenotype linkage

Genotype: DNA/RNA sequence
Phenotype: protein function

You must physically link them, otherwise you can't recover the sequence of a good binder.

🦠 2. Filamentous Phage Basics (p.3–7)

Structure (image pages explained):

Long filament (~1 µm long, ~6.5 nm wide)
Coat proteins:
- pVIII → major coat (2700 copies)
- pIII, pVI, pVII, pIX → ends of phage

👉 The diagrams (p.3–7) show:

A rod-like particle
DNA inside
Proteins forming a “tube”

Important biology:

Infects E. coli via F-pilus
Non-lytic → does NOT kill host (important for continuous production)
ssDNA genome

🔁 3. Phage Life Cycle (p.6 image)

The image shows:

Attachment via pIII to F-pilus
DNA enters bacteria
Replication of phage DNA
Assembly at membrane
Extrusion (not lysis!)

👉 Key insight:

This allows continuous selection cycles without killing cells

🧪 4. Phage Display Concept (p.8–9)

Core mechanism:

Insert foreign DNA into phage coat protein gene (often pIII or pVIII)
Result: protein/peptide is displayed on phage surface

👉 Image (p.9):

Shows peptides sticking out of phage

🔗 Critical:

The DNA inside encodes the displayed protein → genotype = phenotype

⚠️ 5. Limitations of Phage Display (p.10)

Can disrupt phage structure
Reduced infectivity
Large vectors → poor transformation efficiency
Avidity effects (multivalent binding can fake high affinity)

👉 Important nuance:

Multivalent display ≠ true high affinity

🧬 6. Phagemids & Vector Design (p.12–15)

Phagemid (p.12):

Hybrid between:

Plasmid
Phage system

Contains:

Promoter
Signal peptide (pelB)
Tag (myc)
gIII fusion site

Vector comparison (p.14):

Type	Features
Phage vector	Large, multivalent
Phagemid	Smaller, monovalent

pIII vs pVIII (p.15):

Feature	pIII	pVIII
Copies	3–5	~2700
Display	Monovalent	Multivalent
Protein size	Large	Small peptides

👉 Interpretation:

pIII → accurate affinity selection
pVIII → strong signal but less precise

🍞 7. Yeast Display (p.16–18)

Concept:

Protein displayed on yeast cell surface (Aga2 fusion)

Key numbers:

10,000–100,000 copies per cell

Image explanation:

Protein anchored to cell wall
Tagged for detection

🔬 FACS (p.18):

Cells labeled with fluorescent ligand
Sorted by fluorescence

👉 What the diagram shows:

Laser excitation (488 nm)
Charged plates → sorting cells into tubes

👉 Key advantage:

Quantitative selection (affinity tuning)

🦠 8. Bacterial Display (p.19–20)

Concept:

Proteins displayed on bacterial surface

Table (p.20):

Shows:

Different outer membrane proteins used
Applications:
- Vaccines
- Epitope mapping
- Biocatalysis

👉 Insight:

More diverse scaffolds than phage

⚗️ 9. Move to In Vitro Systems (p.21–26)

Why move in vitro?

Avoid cell limitations
Access huge libraries (~10¹³)

Translation diagrams (p.22–25):

🧬 Ribosome structure (p.22):

A site (incoming tRNA)
P site (peptide bond formation)
E site (exit)

Translation steps:

1. Initiation (p.23)

Ribosome assembles
Start codon recognized

2. Elongation (p.24)

Amino acids added
tRNA cycling

3. Termination (p.25)

Stop codon
Protein released

In vitro protein synthesis (p.26):

Cell extracts provide:
- Ribosomes
- tRNAs
- enzymes

👉 Can be:

Prokaryotic or eukaryotic systems

🔗 10. Linking Genotype & Phenotype (p.27–29)

Problem:

No cell → no natural linkage

Solution:

Physically link RNA/DNA to protein

Polysome display (p.28–29):

Image shows:

DNA → RNA → protein
Ribosome holds complex together
Binding selection
Recover mRNA
Amplify

👉 Key idea:

Ribosome acts as bridge

🧬 11. Ribosome & mRNA Display (p.31–36)

Ribosome display:

Protein stays attached to ribosome + mRNA

mRNA display:

Covalent linkage (often via puromycin)

⚠️ Limitations (p.36):

mRNA instability
Requires:
- Low temperature
- High Mg²⁺

🧪 12. DNA-Based Display (CIS display) (p.37–44)

Idea:

Link protein directly to its DNA

Mechanism (p.40–43 images):

RepA protein binds its own DNA (cis-action)
During translation:
- Protein attaches to its encoding DNA

👉 Result:

Stable DNA–protein complex

Selection cycle (p.44):

Incubate with target
Wash weak binders
Elute strong binders
PCR amplify
Repeat

🧫 13. Compartmentalisation (p.46–49)

Concept:

Encapsulate single genes in droplets

👉 Each droplet = mini reaction chamber

Advanced version:

Water-in-oil emulsions
Each compartment:
- DNA
- translation system
- product

👉 Enables:

True genotype–phenotype isolation

🎯 14. Selection Criteria (p.50–51)

You can select for:

Affinity (binding)
Stability
Catalysis

Affinity selection:

Bind to target → keep
PCR amplify binders

✂️ 15. Selection by Proteolysis (p.52–54)

Principle:

Folded proteins → resistant
Unfolded → degraded

Image explanation (p.54):

Folded fusion protein survives → infective phage
Unfolded → degraded → non-infective

👉 Clever proxy for protein stability

🦠 16. Selection by Infection (p.53)

Infection requires pIII domains (D1, D2)
If disrupted → no infection

👉 Used to link:

Structure/function → infectivity

⚗️ 17. Selection for Catalysis (p.55–59)

Concept:

Detect enzyme activity, not just binding

Mechanism (p.55):

Enzyme converts substrate → product
Product is captured (e.g., biotin-streptavidin)

Examples (p.56–58):

Enzyme linked to phage
Reaction generates:
- detectable product
- or capture tag

⚠️ Limitation (p.59):

Single turnover is inefficient

👉 Solution:

Use compartmentalisation → allows multiple catalytic cycles

🧠 Final Big Picture

Core pipeline across all systems:

Generate library (DNA diversity)
Express/display protein
Select based on:
- binding
- stability
- catalysis
Recover genetic material
Amplify (PCR)
Repeat (evolution cycles)

🔥 Key Insights to Remember

Genotype–phenotype linkage is EVERYTHING
Display system choice = trade-off
- Phage → robust, easy
- Yeast → quantitative (FACS)
- In vitro → massive libraries
Multivalency can mislead affinity
Compartmentalisation enables true enzyme evolution

Quiz

Score: 0/31 (0%)

Q0. Why is genotype-phenotype linkage essential in protein selection systems?

Because it allows the selected functional protein to be chemically degraded after screening

Because it ensures that a useful protein variant can be traced back to the DNA or RNA sequence that encoded it

Because it prevents mutations from occurring during PCR amplification

Because it guarantees that all displayed proteins have the same binding affinity

Q1. Which statement best describes the main difference between cell-based and fully in vitro protein selection systems?

Cell-based systems require PCR, while in vitro systems do not

Cell-based systems generally depend on living organisms, while in vitro systems use purified or extracted molecular machinery outside cells

Cell-based systems can only select enzymes, while in vitro systems can only select binders

Cell-based systems always have larger library sizes than in vitro systems

Q2. What is the natural host of filamentous phage such as M13, fd, and f1?

Saccharomyces cerevisiae

Escherichia coli

Human epithelial cells

Bacillus subtilis

Q3. Which M13 coat protein is primarily responsible for infection by adsorption to the F-pilus?

pVIII

pVII

pIII

pIX

Q4. Why is pVIII display usually more suitable for small peptides than for large folded proteins?

Because pVIII is present in thousands of copies and forms the main phage cylinder, so large inserts can disrupt phage assembly

Because pVIII is not exposed on the phage surface

Because pVIII is only expressed inside yeast cells

Because pVIII does not require secretion

Q5. What is one important disadvantage of multivalent phage display?

It prevents PCR amplification of selected clones

It can create avidity effects that make weak binders appear stronger than they really are

It blocks all binding to target proteins

It prevents secretion of phage particles

Q6. Which statement best compares phage vectors and phagemid vectors?

Phage vectors are smaller and usually monovalent, while phagemids are larger and polyvalent

Phage vectors contain no phage genes, while phagemids contain the entire phage genome

Phage vectors clone directly into the phage genome, while phagemids are smaller plasmid-like vectors often used for monovalent display

Phage vectors are only used for yeast display, while phagemids are only used for bacterial display

Q7. In yeast display, why is fluorescence-activated cell sorting useful?

It allows cells to be sorted based on fluorescent signals that reflect display level or ligand binding

It converts yeast cells into phage particles

It removes the need for genotype-phenotype linkage

It chemically attaches mRNA to proteins

Q8. What is a key advantage of yeast display compared with many phage display selections?

Yeast display does not require any DNA library

Yeast display can use FACS to quantitatively sort cells by binding and expression signals

Yeast display only produces one protein copy per cell

Yeast display avoids all post-translational processing

Q9. In ribosome display, what keeps genotype and phenotype linked?

A covalent DNA-protein bond formed by RepA

The stalled ribosome complex containing mRNA and the nascent protein

The bacterial cell wall

A phage coat protein fused to the displayed protein

Q10. Why are low temperature and high magnesium often important in RNA-based display systems?

They increase phage infectivity

They help stabilize mRNA and ribosome or mRNA-protein complexes

They remove stop codons from DNA

They make proteins unfold faster

Q11. What distinguishes mRNA display from ribosome display?

mRNA display usually links protein covalently to its mRNA, often through puromycin, while ribosome display relies on a non-covalent ribosome complex

mRNA display requires living yeast cells, while ribosome display requires phage infection

mRNA display cannot be amplified after selection

mRNA display only works with DNA, not RNA

Q12. What is the central principle of CIS display?

A displayed protein binds only to proteins translated from other DNA molecules

A protein becomes linked to the same DNA molecule that encoded it through cis-acting binding

A phage coat protein is cleaved by trypsin to release DNA

A yeast cell displays antibodies using Aga proteins

Q13. In compartmentalisation-based selection, what is the purpose of water-in-oil droplets?

To create tiny isolated reaction chambers that keep each genotype and its phenotype together

To dissolve phage coat proteins before infection

To prevent proteins from folding

To make all enzymes perform only one turnover

Q14. Why is compartmentalisation especially useful for enzyme engineering?

Because enzymes are selected only by binding strength

Because droplets can confine enzyme, substrate, product, and encoding gene, allowing catalytic activity to be linked to genotype

Because it prevents enzymes from catalyzing more than one reaction

Because it removes the need to recover DNA

Q15. Filamentous phage such as M13 are lytic and kill their E. coli host during phage release.

True

False

Q16. The M13 major coat protein pVIII is present in thousands of copies and forms most of the phage cylinder.

True

False

Q17. pIII display is generally more suitable than pVIII display for larger proteins because pIII is present in only a few copies at the phage terminus.

True

False

Q18. Avidity effects can cause multivalent display systems to overestimate the true affinity of individual binding interactions.

True

False

Q19. Phagemid systems are typically larger than full phage vectors and therefore always have lower transformation efficiency.

True

False

Q20. Yeast display can show high copy numbers of displayed protein on the cell surface, often thousands to tens of thousands of copies per cell.

True

False

Q21. FACS sorting separates cells based on fluorescence signals, which can reflect ligand binding or expression level.

True

False

Q22. In vitro protein synthesis requires intact living cells to grow and divide during the selection process.

True

False

Q23. In ribosome display, removing the stop codon can help prevent release of the nascent protein from the ribosome.

True

False

Q24. mRNA display is generally based on a covalent linkage between the protein and its mRNA.

True

False

Q25. RNA-based selection systems can be limited by mRNA instability.

True

False

Q26. CIS display links phenotype to genotype by coupling the displayed protein to DNA rather than RNA.

True

False

Q27. During a typical CIS display selection cycle, binders are isolated, amplified by PCR, and used for another round of selection.

True

False

Q28. In proteolytic selection for structure, unfolded proteins are often more resistant to protease digestion than folded proteins.

True

False

Q29. Selection by infection can be used when the displayed protein affects whether essential pIII infection domains remain functional.

True

False

Q30. Single-turnover selections are always ideal for enzyme engineering because they strongly amplify catalytic differences.

True

False