Download 蛋白質工程於生物技術 之應用與發展 Protein Engineering

蛋白質工程於生物技術
之應用與發展
Protein Engineering Applications and
Progress in Biotechnology
Protein Functions
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
Proteins

Protein is synthesized via the following processes:
DNA
Gene
RNA
Protein
Function
Each protein is made from the 20
standard amino acids and fold into a
specific 3-D structure.
Proteins
Principle in Protein Biotechnology
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
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 information
 Structure and alignments
Principle in Protein Biotechnology
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
Cloning and expression of target gene:
Gene of Interest
+
Expression Vector
Expression of Fusion Protein
Recombinant
Vector
SDS-PAGE electrophoresis
Protein separation: check purity and MW
Protein Expression Test
I
1
2
3
4
5*
6
(32.2KDa)
(51KDa)
(92.7KDa)
(33.4KDa)
(73.3KDa)
(47KDa)
N
S
I
N
S
I
N
S
I
N
S
I
N
S
I
N
S
116
66
45
35
25
I : cell extract of induction
N : cell extract of no-induction
S : solubility
Principle in Protein Biotechnology
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
Column Chromatography
Protein separation and purification
Ion Exchange
Gel Filtration
Affinity Chromatography
SDS PAGE of Purification
1.
2.
3.
4.
5.
Total proteins
High Salt
Ion exchange
Gel-filtration
Affinity
Principle in Protein Biotechnology
Bioinformatics
Target identification and cloning
Protein expression test
Protein purification and production
Applications
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 function
Applications 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
DNA

RNA
Protein
Irrational engineering (random
mutagenesis) and rational engineering
(site-directed mutagenesis)
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.
In shuffling, the products are degraded to random small
fragments with DNAse I.
x
x
x
DNAse I
x
x
x
x
x
Then full-length sequences are re-assembled
by enzymatic DNA synthesis
Denature, reanneal,
enzymatic DNA synthesis
x
x
x
x
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
THR
SER
HIS
LYS
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
No GFP
Clontech
wt
cycle 2
mutant
cycle 3
mutant
Comparison of the fluorescence of different GFP
constructs in whole E. coli cells
High-throughput screening
Proteins can be engineered using sitedirected 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
a
A)
B)
C)
a
*c
* b
d
*b
*c
*
*
d
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
Computer
Modeling
characterization
Gene
construction
Protein
production
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?
"Fluorescent Timer": Protein
That Changes Color with Time
A fluorescent protein that changes color
with time was generated from the red
fluorescent protein (RFP)
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 vitro
analysis).
E5 used as a
fluorescent
clock:
heat shockregulated
expression of
the E5 mutant
RFP in C.
elegans.