蛋白質工程於生物技術之應用與發展Protein Engineering Applications and Progress in Biotechnology

Structure Structural support: collagen (膠原蛋白 )

Transport Hemoglobin (血紅素 ): transports oxygen from the lungs to cells

Storage Myoglobin (肌紅蛋白 ): stores oxygen in muscles

Hormones Insulin (胰島素 ): protein hormone controls blood glucose level

Enzymes (酵素 )Alcohol dehydrogenase (醇脫氫酶 ) that breaks down alcohols

Protein Functions

Proteins

Protein is synthesized via the following processes:

Each protein is made from the 20 standard amino acids and fold into a specific 3-D structure.

DNA RNA Protein

Gene Function

Proteins

Bioinformatics

Target identification and cloning

Protein expression test

Protein purification and production

Applications

Principle in Protein Biotechnology

Bioinformatics: exploitation of the genome

Bioinformatics is central to the interpretation and exploitation of the wealth of biological data generated in genome projects

Exploitation of the wealth of information from the genomes of human and model organism is critical to biotechnology research

Applications: sequence analysis Search for conserved domains protein structure analysis

E. coli genome

Data sources

NCBI: www.ncbi.nlm.nih.gov National Center for Biotechnology Information

GenBank Files and a relational database with web access

Extensively integrated sequence informationStructure and alignments

Bioinformatics

Target identification and cloning

Protein expression test

Protein purification and production

Applications

Principle in Protein Biotechnology

Cloning and expression of target gene:

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Recombinant Vector

Gene of Interest

Expression Vector

Expression of Fusion Protein

SDS-PAGE electrophoresisProtein separation: check purity and MW

I : cell extract of inductionN : cell extract of no-inductionS : solubility

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45

I N S I N S I N S I N S I N S I N S

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(32.2KDa)

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(51KDa)

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(92.7KDa)

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(33.4KDa)

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(47KDa)

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25

5*

(73.3KDa)

Protein Expression Test

Bioinformatics

Target identification and cloning

Protein expression test

Protein purification and production

Applications

Principle in Protein Biotechnology

Column ChromatographyProtein separation and purification

Ion Exchange

Gel Filtration

Affinity Chromatography

SDS PAGE of Purification

1. Total proteins2. High Salt3. Ion exchange4. Gel-filtration5. Affinity

Bioinformatics

Target identification and cloning

Protein expression test

Protein purification and production

Applications

Principle in Protein Biotechnology

Applications

Functional Studies

Enzymatic Assays

Protein-protein interactions

Protein Ligand Interactions

Structural Studies

Protein Crystallography & NMR Structure Determination

Target Proteins for Rational Drug Design

Therapeutic Proteins – Preclinical Studies

Protein Engineering

Ulmer, K. M. (1983) “Protein Engineering”, Science, 219: 666-671.

Deliberate design and production of proteins with novel or altered structure and properties, that are not found in natural proteins.

Why Engineering Proteins ?

To study protein structure and functionApplications in industry (enzymes) and medicine (drugs)-- New and improved proteins are always wanted.

Example: Extremophilic proteins have been found in nature (temperatures, salt concentrations, pH values) could be useful.

Factors that make the proteins from thermophilic microorganisms more stable

Thermophilic enzymes usually exhibit optimal activity at a higher temperature than the mesophilic enzymes.

No general rules revealed (the best way is to measure experimentally).

General features of the thermostable enzymes:

Increase of compactness and better packing Increase in electrostatic interactions (with the

formation of additional ion pairs) Additional H-bonds Additional disulphide bridges Increasing internal hydrophobic interactions.

Methods for protein engineering

Chemical or Genetic?

Chemical modifications -- in vitro engineering

One of the first way and a re-emerged method for altering protein properties.

Polyethylene glycol (PEG) modification of protein surface amino groups reduces immunoreactivity, prolongs clearance times, improve biostability, increase the solubility and activity of enzymes in organic solvents.

DeSantis, G. and Jones, J.B. (1999) “Chemical modification of enzymes for enhanced functionality”, Current Opinion in Biotechnology, 10(4):324-330

Genetic modification -- in vivo engineering

Genes (DNA) encoding proteins are mutagenized

Irrational engineering (random mutagenesis) and rational engineering (site-directed mutagenesis)

DNA RNA Protein

PCR: Polymerase Chain Reaction

PCR: Polymerase Chain Reaction

Proteins with new properties can be obtained by random mutagenesis

DNA in cells are randomly mutated: chemical mutagens (e.g., hydroxyamine, sodium bisulfite), enzymatic synthesis, mutagenic strains of bacteria (with deficient repairing systems).

Can be applied when the current theories are inadequate to predict which structural changes will give improvement on certain property.

Appropriate procedures for screening or selecting for desired properties are needed

Protein could be made to evolve in vitro

DNA shuffling: in vitro homologous recombination and in vitro protein evolution.

Random mutagenesis by error-prone PCR(with excess of one dNTP) to generate diversity of templates (naturally occurring homologous genes can also be used).

Selection under increasing selective pressures (antibiotics, pH, organic solvent).

Combination with High-throughput screening

DNA shuffling: a method for in vitro homologous recombination between mutant genes.

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DNAse I

In shuffling, the products are degraded to random small fragments with DNAse I.

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Then full-length sequences are re-assembledby enzymatic DNA synthesis

Denature, reanneal,enzymatic DNA synthesis

Some products consist of full-length sequences containing several mutations. Recombinants with improved functions are selected

Example:Development of novel -lactamases with increased activity towards certain substrates.

The -lactam antibiotic cefotaxime is poorly hydrolysed by TEM -lactamase.

Mutant -lactamase genes were shuffled to produce new recombinant genes.

The 1st round of shuffling yielded enzymes conferring resistance to 0.32 - 0.64 g/ml cefotaxime.

Shuffling of these genes yielded enzymes resistant to 5 to 10 g/ml.

A 3rd round of shuffling yielded enzymes resistant to 640 g/ml cefotaxime.

Sequencing of cefotaximeR genes revealed several point mutations.

Six AA replacements were found to confer the high resistance phenotype.

ALA42 GLY92 GLY104 MET182 GLY238 ARG241

GLY SER LYS THR SER HIS

Nature. 1994;370(6488):389-91

One more example: Improved GFP was generated by DNA shuffling

Started with a synthetic GFP gene Performed recursive cycles of DNA shuffling. Screened for the brightest E.coli colonies (using UV light). After 3 cycles of DNA shuffling, a mutant was obtained with

45-fold greater fluorescence.

Nature Biotechnology, 1996, 14:315-319

Comparison of the fluorescence of different GFP constructs in whole E. coli cells

No GFP Clontech wt cycle 2 mutant

cycle 3 mutant

High-throughput screening

Proteins can be engineered using site-directed mutagenesis

Nucleotide residues to be mutated need to be first identified: by using information from 3-D structure, homology comparison, and etc.

Nucleotide and Amino acid residues can be replaced, deleted or added.

PCR technology can be used to carry out site-specific mutagenesis

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Applications in Engineering Proteins

•Engineering of industrial enzymes

•Re-design of substrate specificity

•Folding and stability

•Custom-designed proteins

•Chimeric protein constructions

Novel proteins may be generated by de novo design

ComputerModeling

Gene construction

Protein productioncharacterization

De novo design of proteins: The attempt to choose an amino acid sequence that is unrelated to any natural sequence, but will fold into a desired 3-D structure with desired properties.

Interesting Examples?

A fluorescent protein that changes color with time was generated from the red

fluorescent protein (RFP)

"Fluorescent Timer": Protein That Changes Color with Time

Science, 24 November, 2000, Vol.290:1585-1599.

The RFP gene was mutated by error-prone PCR. Mutants exhibiting a green intermediate fluorescence were

screened visually by using a fluorescent microscope. Mutants with various properties, such as faster maturation, double

emission (green and red), or exclusive green fluorescence were isolated.

One mutant protein (E5) changes color over time: initially bright green, then to yellow, orange, and finally red.

E5 has two replacements: V105A and S197T.

Time course of green and red fluorescence in E5 RFP (in vitroanalysis).

E5 used as a fluorescentclock:heat shock-regulatedexpression ofthe E5 mutantRFP in C.elegans.