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MindMap Gallery protein engineering

protein engineering

This is a mind map about protein engineering, full of useful information, interested friends can refer to it!

Edited at 2023-11-24 01:39:34

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protein engineering

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Outline

2023 Medical AI Industry Research
- 20
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protein engineering

protein

Physiological function

Catalytic function

enzyme

Adjustment function

hormone

structural function

Cytoskeleton

Transport function

hemoglobin

Immune Function

Immunoglobulin

Motor function

flagella, muscle protein

Storage function

Casein

Biofilm function and nerve conduction, etc.

importance in human life

Basic substances necessary to sustain life.

Using proteins to diagnose and treat certain diseases.

The food industry uses proteins to manufacture a variety of products.

Limitations of practical applications

To function: under physiological conditions

protein engineering

concept

On the molecular structure of existing proteins or on the genes encoding proteins → Transform or newly design proteins → Obtain new protein products suitable for human needs.

Food protein modification technology

Functional properties of proteins

Hydration properties

Solubility

Dispersion

Swellability

Thickening

Emulsifying properties

emulsifying

Foamability

water holding capacity

Oil retention

Rheological & Textural Properties

Gellability

Adhesion

elasticity

concept

Chemical, physical, biological (enzyme) means → essentially change the functional properties of protein → the quality properties required for food.

method

chemical modification

Targeting: amino, hydroxyl, sulfhydryl & carboxyl groups → chemical modification → changing protein structure, electrostatic charge, hydrophobic groups → changing the functional properties of the groups.

Enzymatic modification

Enzymatic hydrolysis modification

Protease → degrade: food protein → improve: solubility, dispersion, emulsification.

Enzymatic polymerization modification

Transglutaminase → protein: polymerization modification → improve the functional properties of food proteins.

Physical modification

Various physical field effects → change the functional properties of proteins.

Textured extrusion modification

High voltage electrostatic modification

High heat and high pressure modification

Ultrasonic modification

High frequency electric field modification

Microwave modification

…

technical means

protein level

chemical modification

Enzymatic hydrolysis or polymerization modification

Physical modification

Modify existing proteins

gene level

Rational molecular design & site-directed mutagenesis

rational molecular design

concept

On the basis of the known three-dimensional structure and function of a protein, targeted mutations are carried out on a gene sequence that is most likely to affect the function and properties of the protein, and one or two amino acid residues or modules of the protein are purposefully changed to construct a new protein molecule. .

The basic steps

Isolate and purify the target protein, crystallize it, and learn as much information as possible about its spatial structure.

Determine its functional domain.

Analyze the interrelationship between structure & function and find out the key structure & groups.

Focusing on these key groups & structures → propose a plan to modify the protein & use genetic engineering methods (sited mutation) → implement it.

For modified proteins → functional assay, site-directed mutagenesis.

Positioned mutagenesis

concept

According to the rational molecular design scheme → the DNA sequence encoding the protein is known → substitution, insertion or deletion: the selected nucleotide.

Commonly used techniques

Oligonucleotide primer-mediated site-directed mutagenesis

principle

Oligonucleotide fragment containing mutated base → Primer → Polymerase action → Start DNA molecule → Replication

include

Kunkel mutation method

Antibiotic-based “resistance recovery” mutation method

Mutation method based on removal of specific restriction enzyme cutting sites

Advantages and Disadvantages

advantage

Higher fidelity than recombinant PCR mutation method

shortcoming

The operation process is complex and the cycle is long

Cloning of the gene to be mutated will be restricted by restriction enzyme sites

PCR-mediated site-directed mutagenesis

principle

PCR → insertion & deletion of mutated bases → primers → two pairs of primers → nucleic acids: PCR amplification → overlapping extension → two PCR products with partially overlapping sequences → mixing, denaturation, renaturation and chain extension → one pair and two Complementary primers on the outside of the fragment to be spliced → second amplification → full-length heterologous hybrid double-stranded DNA.

Advantages and Disadvantages

advantage

easy to use

Mutation rate 100%

shortcoming

Fidelity is low

Follow-up work is complicated

cassette mutation

principle

Also known as: fragment substitution method

Target gene sequence: appropriate restriction enzyme site → insertion: various suitable mutated DNA fragments → substitution: specific DNA fragment in the target gene.

include

cassette substitution mutagenesis

Mixed oligonucleotide mutagenesis

Advantages and Disadvantages

advantage

Simple and easy

High mutation efficiency

Change multiple sites or one fragment at a time

shortcoming

Synthetic DNA fragments are expensive

Requires appropriate restriction enzyme sites

Application: Enzyme structure modification

Enhance stability↑

Improve enzyme activity↑

Change enzyme selectivity

Eliminate the antioxidant properties of enzymes

Introduce disulfide bonds

Convert amino acid residues

Improve protein thermal stability

Change the pH conditions optimal for the enzyme

Cassette mutation → glucose isomerase molecule

Modify the catalytic specificity of the enzyme

Site-directed mutagenesis technology→glucoamylase

directed evolution in vitro

Irrational molecular design

Theoretical source

Utilize genetic engineering principles → Laboratory: simulate the biological evolution process.

concept

In vitro directed evolution of proteins: also known as molecular evolution,

That is: laboratory conditions → simulate the natural evolution mechanism: mutagenesis and recombination of genes encoding proteins → high-throughput screening → select proteins with better performance.

Not required: Structural information about the protein is known

Random mutation directional selection = target mutant

Vs natural evolution

Different evolutionary dynamics

Different directions of evolution

Different rates of evolution

directed evolution

Mutation sites: random, uncertain, & number: uncertain

Mutational effects: unpredictable

fixed point evolution

Mutation site: determined, & number: predicted

Mutational effects: probably known

How to obtain random mutants (Commonly used directed evolution technology)

Reorganization

DNA shuffling

A group of closely related DNA sequences → DNaseI → Random digestion → Many fragments → Partially overlapping base sequences → Self-guided PCR recombination → Full-length genes → Diversity library → Screening of mutation libraries → Improved mutants → Next round of templates → Repeat Multiple rearrangements and screening → Obtain mutants with satisfactory characteristics

principle

Exon shuffling

Random in vitro priming shuffle

Synthesis Reorganization

mutation

fault-tolerant PCR

Taq polymerase → PCR amplification of the target gene, while introducing base mismatch → random mutation of the target gene

principle

Randomly positioned mutations

Staggered extension

…

Application: Enzyme preparation transformation

L-aspartase: Improve enzyme heat resistance & enzyme activity

Phospholipase A1 mutants: resistant to organic solutions

Hydantoinase: D-form substrate mutates to L-form substrate

Fusion protein technology

concept

Purpose → connect two/multiple gene coding regions that encode functional proteins together → end to end → the same regulatory sequence: control the composed gene expression product → express the required protein.

method

Formation of PCR-Mediated Protein Molecular Chimeras

Intron-mediated formation of protein molecular chimeras

Ligase direct ligation

application

bifunctional enzyme

Retain the respective enzymatic activities of the constituted enzyme molecules

Can produce: "proximity effect"

Targeted drugs/directed drugs

composition

drug

Ligand that can bind specifically to lesions

Can form: proteins with unique ideas & functions

Antimicrobial peptides

Peptide antibiotics

Naturally produced by the body / an important component of innate immunity

Powerful antibacterial effect against drug-resistant bacteria

A class of cationic small molecule peptides with broad-spectrum antibacterial activity

New protein design

concept

According to the desired protein structure & function → design the amino acid sequence,

Name: anti-folding research

Need to understand: the relationship between protein structure & function

The basic steps

Propose basic structural drawings

Determine the amino acid sequence

Structural optimization

progress made

Create new proteins

Protein engineering at the genetic level three levels

primary renovation

Changes in individual amino acids

Deletion, substitution or insertion of an entire amino acid sequence

Advanced makeover

Tailoring of protein molecules

Such as: domain splicing, fusion protein technology

Design and synthesize new proteins from scratch

List 3 examples of protein engineering applications in food

Introduce disulfide bonds → improve the thermal stability of proteins

Convert amino acid residues → improve protein thermal stability

Change the optimal pH value of the enzyme → increase the lysine content in corn