Protein Biotechnology

(1)

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

(2)

Manifestation of Novel Social Challenges of the European Union

in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

Identification number:

TÁMOP- 4.1.2-08/1/A-2009-0011

Title:

PROTEIN BIOTECHNOLOGY Leader:

Dr. József Tőzsér

(3)

BIOCHEMICAL PROPERTIES OF PROTEINS. PROTEIN SYNTHESIS.

REVIEW OF THE DIFFERENCES

BETWEEN THE EUKARYOTIC AND PROKARYOTIC PROTEIN

SYNTHESIS

Éva Csősz

Protein Biotechnology - Lecture 1

(4)

Lysine Leucine

Isoleucine Histidine

Serine Proline

Phenilalanine Methionin

Threonine Triptophane Tyrosine Valine Glutamate

Glutamine

Cysteine Glycine

Arginine Asparagine Aspartate Alanine

The protein constituent amino acids

(5)

The structure of globular proteins

(6)

Anionic Cationic Amfoter Non ionic Detergents:

Classification of detergents according to their charge

(7)

PM IC EC

aggregate

+

A. B.

The effect of detergents on protein structure

(8)

S S

Protein SH

SH Protein

SDS Urea Thiourea

β-mercaptoethanol DTT

TCEP S

S Protein

S S Protein

SH SH Protein

SDS Urea Thiourea

+

A.

B.

The effect of reducing agents on protein structure

(9)

A. B. C.

The structure of tRNA

(10)

Met

Aminoacil-tRNA ATP

Met-tRNA synthase

Met-AMP

+ PPi

tRNA^Met

1.

2.

AMP

The activation of amino acids. The charging of aminoacil-tRNA

(11)

5’ UTR 5’

3’

3’ UTR Start codon

(AUG)

Stop codon (UAG, UAA, UGA) Coding region

Ribosome binding site

Shine-Dalgarno sequence Shine-Dalgarno sequence: AGGAGGxxAUG

Stop codon (UAG, UAA, UGA) Start codon

(AUG)

5’ UTR 3’ UTR AAAAAA 3’

5’ m⁷G

Kozak sequence

5’ cap poliA tail

The prokaryotic mRNA

The eukaryotic mRNA

Kozak sequence: Gcc A ccAUGG G

Ribosome binding site

Coding fMet

Coding Met

Coding region

The structure of prokaryotic and eukaryotic mRNA

(12)

Shine-Dalgarno sequence

AUG AUG AUG AUG

Promoter Operator Gene 1 Gene 2 Gene 3

Shine-Dalgarno sequence Shine-Dalgarno sequence

fMet fMet

fMet Met

DNA

mRNA

protein

5’ 3’

AAAAAA m⁷G

Gene

DNA mRNA

protein

AUG AUG

Met Met

The differences between the prokaryotic and eukaryotic mRNA

B.

A.

(13)

50S big subunit 23S RNA 5S RNA 35 protein

30S small subunit 16S RNA 21 protein 70S ribosome

The prokaryotic ribosome (70S)

60S big subunit 28S RNA

5S RNA, 5.8S RNA 49 protein

40S small subunit 18S RNA 33 protein 80S ribosome

The eukaryotic ribosome (80S)

The structure of ribosomes

(14)

mRNA

40S small subunit 60S big subunit

A site - for aminoacil-tRNA binding

P site – for tRNA binding

A P

5’ E 3’

E site – for the release of the empty tRNA

Direction of translation

Nascent polypeptide chain The structure of the active ribosome

(15)

5’ UTR 3’ UTR 3’

5’

Shine-Dalgarno sequence

AUG

fMet IF-2

30S subunit IF-3 IF-1

The initiation of protein synthesis in prokaryotes

(16)

5’ m⁷G

Kozak sequence

AUG

Met

eIF-2 40S subunit

The initiation of protein synthesis in eukaryotes

(17)

40S subunit

5’ m⁷G

AUG

Met

eIF-2

Met GTP

eIF-2 GDP

60S subunit

The end of initiation

(18)

A P

E

A P

E E P A

A P

E

Met

EF-Tu GDP

EF-Tu GTP

Ala

EF-Tu GTP

Met Ala Ala

The entry of the new aminoacil-tRNA

Met Ala

Formation of the new peptide bond (Peptidyl

transferase)

Translocation

Met Ala

EF-G EF-G

GTP GDP

mRNA

mRNA mRNA

mRNA

5’ 3’

5’

3’

The second step of protein synthesis: the elongation

(19)

mRNA

+NH₃

5’ 3’

Direction of translation

Direction of peptide chain growth

The major features of elongation

(20)

mRNA

+NH₃

5’ UAG 3’

STOP codon

RF1

mRNA

5’ UAG 3’

RF1

COO^-

+NH₃

The third step of protein synthesis: the termination

(21)

DNA mRNA

mRNA nucleus

cytosol

+NH₃

The localization of protein synthesis in eukaryotes

(22)

The localization of protein synthesis in prokaryotes

(23)

PROTEIN FOLDING, 3D

STRUCTURE FORMATION.

CHAPERONS. FOLDING PROBLEMS, FOLDING DISEASES

Éva Csősz

Protein Biotechnology - Lecture 2

(24)

Hydrogen donor Hydrogen acceptor

– N – H – N – H – O – H – O – H

N – O – N – O –

3,04 Å 2,88 Å 3,1 Å

2,7 Å

Hydrogen bond

Covalent bond

Strong hydrogen bonds

– O – H

O

Weak hydrogen bond

The most abundant bonds in the living system: hydrogen bonds

(25)

R – COO ^- ^{2,8 Å} ⁺ H₃N – R A.

B.

The most abundant bonds in the living system: electrostatic interactions

(26)

apolar molecule

A. B.

The most abundant bonds in the living system: hydrophobic interactions

(27)

+ - - -

+

- -

- + +

+ + +

- + + +

-

+ +

+ - -

- - -

A.

B.

The polar water molecules stabilize the structures in the living systems

(28)

Protein folding

(29)

Primary structure Amino acid

sequence

Secondary structure Local structures

Tertiary structure The 3D structure of

the polypeptide chain

Quaternary structure The structure of proteins

consisting of several polypeptide chains

The structure of proteins

(30)

COO^-

+NH₃

S S

N-terminus

C-terminus

The primary structure of proteins: the amino acid sequence

(31)

The peptide bond

(32)

A. B.

The alpha helix

(33)

Beta sheet

Beta turn

The beta sheet

(34)

Hydrophobicity scale

Hydrophobic amino acids inside, hydrophylic ones outside

The prediction of protein structure based on the position of hydrophobic amino acids

(35)

Secondary structure

Modules

Tertiary structure

The tertiary structure of the proteins is made up of secondary structure elements

(36)

RNase

Native form (100 % activity)

Denatured form (0 % activity)

Scrambled form (1-2 % activity) Denatured form

(0 % activity)

Urea removal Oxidation

Oxidation Urea removal Reduction

Denaturation (8M urea)

RNase RNase

RNase

RNase RNase

Reduction Denaturation (8M urea)

The Anfinsen experiment

(37)

Folding funnel

Free energy decreases The number of possible conformation decreases

The folding of proteins through metastabile intermediates

(38)

Intrinsically disordered region

Subunit 2 Subunit 1

The structure of intrinsically disordered proteins

(39)

The intrinsically disordered proteins can adopt alpha helix or beta sheet structure upon interacting with other proteins

(40)

The crystal structure of the GroEL chaperon

(41)

Chaperon

Misfolded structure

Native structure

The function of chaperons

(42)

The role of chaperons in the formation and maintenance of the protein 3D structure

(43)

Hsp 20-30 oligomers

Hsp 60 oligomers

Hsp 70 monomers

Hsp 90 dimers

Hsp 110 oligomers

Classification of chaperons according to their structure

(44)

Anfinsen cage

ATP

ADP

ADP ADP ADP

ATP

ATP ATP

ADP

hsp10

hsp60

15 s

Native protein Misfolded

protein

The function of Hsp60

(45)

(46)

HOP Hsp70

Hsp40

Hip

HOP

Hip SHR

SHR

Hsp90

Hsp70

Hsp40

Hip HOP

SHR

SHR SHR Hsp90

Hsp90

Hormone Foldosome

Immature receptor–

unable to bind hormones

Mature receptor – able to bind

hormones DNA binding domain

DNA binding

Immunophilin – peptidyl- prolyl cis-trans isomerase p23

p23

ATP

(47)

Hsp 110

ATP

hsp70 Native protein

Denatured protein

Aggregated proteins

Native protein The function of Hsp110

(48)

The function of calnexin and calreticulin

(49)

S S

S S PDI SH

PDI SH SH

HS-

PDI SH SH

1st conformation 2nd conformation

The function of protein disulfid isomerase (PDI)

(50)

cis Pro trans Pro

The function of peptidil-prolyl isomerase

(51)

Native state Abnormal state

Entropy

Free energy

The appearance of proteins with abnormal structure during the folding process

(52)

Native, endogenious PrP^c

Spontaneous appearance of PrP^sc

Interaction of PrP^sc with endogenious

PrP^c-vel

Interaction of PrP^sc with endogenious

PrP^c-vel

The number of „bad” prions increase upon coming in contact with native, endogenious forms

(53)

Native molten globule

denatured

aggregation

Amyloid fibers

Amyloid plaques

Native Amyloid fibers

molten globule

The probable mechanism of amyloid plaque formation

(54)

PROTEIN SORTING AND TARGETING

Éva Csősz

Protein Biotechnology - Lecture 3

(55)

5’ 3’

2.

3.

4.

5.

cytosol

ER lumen SRP receptor

SRP

mRNA

ER membrane

1.

Protein targeting to endoplasmic reticulum I.

(56)

SRP receptor

ER lumen

SRP ER membrane

Cytosol

5. 7.

Signal peptidase

5’

3’

mRNA

6.

Protein targeting to endoplasmic reticulum II.

(57)

SRP receptor

ER lumen

SRP ER membrane

3’

mRNA 5’

8.

9.

10.

Cytosol

UAA

Protein targeting to endoplasmic reticulum III.

(58)

Protein targeting to endoplasmic reticulum and their cotranslational modification

(59)

The cotranlational N-glycosidation of proteins in the endoplasmic reticulum

(60)

P

N-Acetil glucosamine Mannose Glucose

Dolichol-phosphate

P

P P

5

4

P P

3

Reorientation Cytosol

ER lumen ER membrane

AsnXxxSer/Thr

oligosaccharide chain on dolichol phosphate

(61)

U U

U U U Misfolded

proteins

Properly folded proteins

Grp78

PDI ERAD chaperon

ER lumen

Cytosol

Proteosome

Protein degradation

Quality control in the endoplasmic reticulum

(62)

Plasma membrane

Lysosome

Extracellular space

Nucleus

Cytosol Nucleus RER

Cis Golgi network

Trans Golgi network Golgi cisternae

Cytosol

Secretory vezicle 2.

4. 5.

6.

3.

7.

1.

The route of proteins among different compartments

(63)

P P P P

P

N-Acetil glucosamine Mannose Glucose

The modification of proteins in the Golgi compartments

(64)

Mannose rich type

Complex type

N-Acetil glucosamine Mannose Glucose Galactose Sialic acid Lysosome

Extracellular space/plasma membrane

In Golgi apparatus different N-glycosilated proteins are formed with various sugar composition

(65)

N

N C

C

C Hsp

70

mHsp 70

Mitochondrial matrix

Outer membrane

Inner membrane Import receptor

Protein targeting to the mitochondrium

(66)

Protein targeting to the nucleus

(67)

STUDY OF PROTEIN STRUCTURE: X-RAY CRYSTALLOGRAPHY, NMR, MASS

SPECTROMETRY

Éva Csősz

Protein Biotechnology - Lecture 4

(68)

Prediction of intrinsically disordered regions based on their sequence -

IUPred (iupred.enzim.hu)

Protein crystal structure

The relation between protein structure and the presence of intrinsical disorder

(69)

High energy X-ray

The X-ray scatters on the electrons of the protein crystal and a specific diffraction map is generated.

Protein crystallization

Suitable crystals

Determination of protein structure with X-ray crystallography I.

(70)

Diffraction map

Electron density map

(administrating Fourier transformation)

Modelled structure

Determination of protein structure with X-ray crystallography II.

(71)

5 Å 3 Å 1,5 Å

The quality of amino acid residue fitting into the electron density map determines the quality of the crystal structure

(72)

Determination of protein structure with nuclear magnetic resonance (NMR)

(73)

Protein Data Bank (PDB) – the repository of determined protein structures

(74)

Electronics

Vacuum system

Mass spectrum Data analysis Detector

Mass analyzer Ion source

Sample

The structure of mass spectrometer

(75)

+20-30 kV

AH⁺ Laser Sample

Sample plate

+

+ + + +

Mass analyzer Detector Matrix

The theory of MALDI – Matrix Assisted Laser Desorption Ionization

(76)

+ + +

++ +

2-4 kV

LC

capillary

+

+ + + +

Mass analyzer Detector + +

+ + + +

+

+ + + + + +

+ Curtain gas Heated orifice

The principle of electrospray ionisation (ESI)

(77)

Ion source Mass analyzer Detector

The ion path in the Time-Of-Flight (TOF) mass analyzer

(78)

Reflectron detector

Improvement of mass resolution of mass spectrometers by the administration of a reflectron

(79)

Quadrupole Detector

The ion path in the quadrupole

(80)

Detector Ion trap

The ion path in the ion trap

(81)

m/z m/z

Ms ms spektrum

AVL/ITL/IDKK

Q1 Collision cell Q3

ESI ion

source Detector

The determination of amino acid sequence using electrospray ionization tandem MS (ESI MS/MS)

(82)

Ion source

Detector IMS T-wave

Intenzitás

Drifting time

Intenzitás

Arrival time (ms) (GRGDS)²⁺

211,7 Å² (SDGRG)²⁺

222,7 Å²

The ion path in the high definition mass spectrometers (HDMS)

(83)

PROTEIN PURIFICATION (CHROMATOGRAPHIC

TECHNIQUES) AND ANALYSIS (SDS-PAGE, 2DE, MASS

SPECTROMETRY)

Éva Csősz

Protein Biotechnology - Lecture 5

(84)

1. Extraction

2. Isolation, concentration, stabilization

3. Removal of bigger impurities

4. Removal of smaller impurities

Number of steps Purity

Points to be considered in chosing the optimal protein purification procedure

(85)

The proteins specifically bind to the column

The unbound proteins are washed away

The elution of the specifically bound

proteins

Purification of proteins using affinity chromatography

(86)

Solution Solution Solution Solution

Separation of proteins with analytical gel filtration

(87)

Desalting of proteins with dialysis

(88)

-

+

Direction of migration

SDS-polyacrylamide gel electrophoresis

(89)

pH 3 pH 10

Isoelectric focusing of proteins

(90)

Mw

(91)

pH 3 pH 10 4%

18%

SyproRuby staining

Ag staining Coomassie stainig

The visualization of proteins with different staining methods

(92)

+

Y

The antibodes specifically bind to the sample

Wash

Y Y Y

Y

Y Y Y

Y Y

Wash Direct IP

Indirect IP Protein A/G

beads

Centrifugation Ag-Ab bond release

Purification of proteins using immunprecipitation (IP)

(93)

Blotting Addition of a specific

antibody PVDF membrane

Protein analysis with Western blot

(94)

Excision of spots

containing proteins

Enzymatic (trypsin) digestion

Mass spectrometry analysis Lysis of the cell

2DE

Protein identification

The visualization of proteomics workflow

(95)

2DE H. influenzae

(control)

H. influenzae (aktinonin treated)

Mixing the samples Labeling with DIGE labels

The analysis of quantitative and qualitative differences using difference gel electrophoresis (DIGE)

(96)

Culture medium with

13C containing Arg (heavy Arg)

Cell lysis and homogenization

Mixing the sample

intensity

m/z

MS/MS

6 Da

Cell lysis and homogenization Culture medium with

normal Arg (light Arg)

Metabolic labeling with SILAC – stable isotope labeling with amino acids in cell culture

(97)

Group responsible for protein binding Balance

group Label group

(114, 115, 116, 117)

Isobaric group

The structure of the iTRAQ label

(98)

Control

Treated cells 60 min Treated cells

120 min Treated cells

240 min 114

115 116 117

Mixing the

sample MS _int^e

nzitás

m/z

intenzitás

m/z

114 115

116

117 MS/MS

Protein content based on area under the curve

Chemical labeling with iTRAQ (isobaric tag for relative and absolute quantitation) technique

(99)

m/z

Q1 Q3 Detector

m/z

Complex protein mixture Fragmentation of parent ions with specified m/z

Selection and detection of

fragment ions with specific m/z ESI ion

source Collision cell

Detection of specific proteins using multiple reaction monitoring

(100)

POSTTRANSLATIONAL

MODIFICATION OF PROTEINS AND THEIR ANALYSIS USING PROTEOMICS METHODS

Éva Csősz

Protein Biotechnology - Lecture 6

(101)

+

HbA1c

Glucose

Glucose Glucose

Glycation

(102)

Tyr kinase

Ser/Thr kinase Thr Ser

Kinase

Phosphatase

ATP ADP

Pi Tyr phosphatase P

Ser/Thr phosphatase

Tyr

The modification of proteins by phosphorylation and dephosphorylation

(103)

3

Farnesylation Geranylation

The modification of proteins by prenylation

(104)

Palmitic acid Myristic acid

The modification of proteins by fatty acid modifications

(105)

Limited proteolysis Degradation

The modification of proteins by proteolysis

(106)

Collagenase

Proteosome

Thrombin Presenilin

Cathepsin G Signalase

The site of proteolytic cleavage

(107)

The effect of posttranslational modifications on gene transcription

(108)

Gln Lys

TG2

Ca²⁺

Formation of isopeptide bonds in the transglutaminase catalyzed reaction

(109)

Sypro Ruby Stains all proteins

ProQ Diamond

Stains only phosphoproteins ProQ Emerald

Stains only glycoproteins

Specific staining procedures used for the detection of posttranslational modifications

(110)

Ser – P Enrichement

(TiO2, IMAC) Ser – P Ser – P Ser – P

Phosphoproteins

PO₃^-

79 Da

98 Da

H₃PO₄loss

CID

The fate of phosphate groups of proteins during mass spectrometry analysis

(111)

m/z

Q1 Q3 Detector

m/z 79

ESI ion

Precursor ion scan

(112)

m/z

Q1 Q3 Detector

m/z ESI ion

Neutral loss scan

(113)

ESI ion source

m/z

Q1 Collision cell Q3

m/z

653 555

Detector

The study of posttranslational modifications using multiple reaction monitoring (MRM)

(114)

THE STUDY OF PROTEIN-PROTEIN INTERACTIONS

Éva Csősz

Protein Biotechnology - Lecture 7

(115)

Hubs

Interaction map

(116)

Protein complex

Antibody against one of the protein in the complex

Y

LC-MS/MS analysis of the peptides

Immunoprecipitation

Trypsin digestion

SDS-PAGE

Identification of the interaction partners

Excision of bands and in-gel digestion

LC-MS/MS analysis of the peptides

The study of protein-protein interactions with co-immunoprecipitation

(117)

Protein complex

Antibody against X

IP

SDS-PAGE

Western blot

Antibody against X

Antibody against Y

The study of protein-protein interactions with pull-down technique

(118)

Membrane

Immobilized protein X

+ Y protein

Y

+ Antibody against Y

Developing

The study of protein-protein interactions with far-Western technique

(119)

Dithiobis-sulfosuccinimidyl-propionate

(120)

Bis-sulfosuccinimidyl-suberate

(121)

Photo-Leu Photo-Met containing medium

Croosslink formation between residues in close proximity

(interaction partners)

Further analysis of the crosslinked proteins Identification of the interaction partners

UV

The study of protein-protein interactions with photoactive crosslinking agents

(122)

Gal4BD domain Gal4AD domain

lacZ

lacZ X

X X

X Y Y

BD

Y

BD

Y BD

+

The study of protein-protein interactions with yeast two hybrid system

(123)

Direct method Indirect

method

Y

Y Y Y

Detection of spots/loci with positive signal.

Antibody chip

Y Y Y

Y

Antibody chip

Glass surface Glass surface

Glass surface

The study of protein-protein interactions using protein chips

(124)

SELDI plate

+

+ + + +

Mass analyzer Detector

MALDI-TOF

Laser Mixing with matrix

Protein chip

Complex protein mixture

The study of protein-protein interactions of immobilized proteins on the surface of the protein chip using SELDI technique

(125)

matrix Binding of specific

phages

Specific elution and phage amplification

The phage display technology

(126)

X Y

YFP CFP

X ^YFP Y ^CFP h 436 nm

480 nm

FRET h 436 nm

535 nm 480 nm Donor

group

Acceptor group

The study of protein-protein interactions with FRET – fluorescence resonance energy transfer

(127)

Y Y Y

Sample

Prism Sensor chip

Gold layer

Flow cell Light

P-polarized light

Reflected light

Detector

Y

Surface plasmon

Immobilized ligand

The study of protein-protein interactions with surface plasmone resonance

(128)

Y Y Y Y Y Y Y Y

Y Y Y Y

time (s) Resonance

signal (kRU)

300 600

0 18

12 Association

Dissociation

Regeneration

k_a

k_d

Concentration

Y Y

k_a k_d

x x

The study of protein-protein interactions with Biacore based on surface plasmone resonance

(129)

HETEROLOGOUS EXPRESSION I

Tamás Emri

Protein Biotechnology - Lecture 8

(130)

Common structure of expression vectors

gene for expression (cDNA, cDNA with artificial intrones, cDNA with/without signal

sequence or tag sequence, cDNA with altered codons, etc.)

sequences for efficient

transcription and translation (e.g. promoter, ribosomal binding site, transcription terminator sequences)

E. coli marker gene (shuttle vectors)

E. coli origin of replication (shuttle vectors)

marker gene working in expression cells (replicative vectors)

origin of replication working in the expression cells (replicative vectors)

other sequences (e.g. genes for stabile replication or integration of the vector, gene for

regulating the promoter, etc.)

(131)

Role of RNA I and RNA II in the replication of plasmids

RNA II

ori

replication

RNA I

rop

no replication

tRNA

enhanced replication

(132)

Integration based on levan sucrase selection

double recombination single recombination

integration of the whole plasmid integration of the green cassette

integration cassette

(133)

HETEROLOGOUS EXPRESSION II

Tamás Emri

Protein Biotechnology - Lecture 9

(134)

Comparison of the Sec and the Tat secretory pathways

translation translation and folding cytosol

extracellular side

Sec folding Tat

(135)

Mechanism of the Sec-pathway

secA

secE secY secG secDF

yajC

ftsY b- SRP

SPase

cytosol

extracellular side

(136)

Structure of the S. carnosus expression cassette

promoter SP pro gene CWA

(137)

Representative N-glycoside oligosaccharides of Saccharomyces cerevisiae and Pichia pastoris

Asn

core-type (S. cerevisiae) 2 GlcNac and <15 Man

Asn

P

n x

mannan-type (S. cerevisiae) 2 GlcNac and 50 < Man

Asn

b-1,4-N-acetyl glucosamine b-1,4-mannose

a-1,3-mannose a-1,6-mannose a-1,2-mannose a-mannose

P. pastoris

(138)

Artificial chromosomes – the alternative of integration

genes (supplemented with promoters and other

sequences needed for efficient transcription and translation)

telomeres (for linearization of the vector, sequences between the two telomeres are eliminated)

centromere

origin of replication, marker gene, shuttle vector sequences etc.

(139)

HETEROLOGOUS EXPRESSION III

Tamás Emri

Protein Biotechnology - Lecture 10

(140)

Position-mediated expression enhancement – functionalized cells

A

B

C

(141)

Representative plant N-glycoside oligosaccharides

Asn

a-1,3-mannose a-1,6-mannose a-1,2-mannose

b-1,2-N-acetyl glucosamine a-1,3-fucose

b-1,2-xylose b-1,3-galactose a-1,4-fucose

Asn

oligosaccharide core developed in ER:

Asn Asn

Asn

(142)

Parts of Agrobacterium tumefaciens Ti plasmid

T DNA

(143)

Transformation with Agrobacterium tumefaciens

plant cell

Agrobacterium

T-DNA

(144)

Representative insect N-glycoside oligosaccharides

Asn

a-1,3-mannose a-1,6-mannose a-1,2-mannose

b-1,2-N-acetyl glucosamine a-1,3-fucose

a-1,6-fucose

Asn Asn

oligosaccharide core developed in ER:

(145)

Integration of expression cassette into the bacmid

bacmid

E. coli

LacZ gene - Tn7 target sequence – LacZ promoter Tn7L and R sequences

donor plasmid

helper plasmid encoding the transposase

bacmid E. coli

donor plasmid

expression cassette integrated into the bacmid

1.

2.

helper plasmid encoding the transposase

(146)

PROTEIN ENGINEERING

Tamás Emri

Protein Biotechnology - Lecture 11

(147)

Impact of de novo protein design

(148)

Structure of a protein developed by de novo protein design

Helix: -Gly-Glu-Leu-Glu-Glu-Leu-Leu-Lys-Lys-Leu-Lys-Glu-Leu-Leu-Lys-Gly Loop: Pro-Arg-Arg

helix

helix-loop-helix

final structure

NH₂-Met-helix-loop-helix-loop-helix-loop-helix-COOH

(149)

Receptor tyrosine kinase (RTK) – mechanism of action

P

P P

P

membrane

RTK

intracellular signaling ligand

(150)

Synthetic growth factors

receptor binding module linker region oligomerization scaffold

oligomer (dimer)

(151)

Development of receptor-specific peptide hormones

A

B

(152)

PCR based site directed mutagenesis

deletion point mutation insertion

sequence for deletion point mutation

in the primer non-complementer loop in the primer

(153)

Directed evolution

gene

wild-type protein

library of mutated genes

library of mutated proteins

screening/selection positive variants repeat

(154)

DNA Shuffling 1

digestion with restriction endonuclease/endonucleases

ligation

gene 1 gene 2

recombinant genes

(155)

DNA Shuffling 2

partial digestion with DNase

PCR without primers mix, de- and renaturation of fragments

PCR with primers designed for the original ends of the genes

gene 1 gene 2

(156)

Staggered extension process (StEP)

A

B C

D

E

(157)

Exon shuffling

homologue genes

recombinant genes containing domains of different origin

domains bordered with different sequences

(158)

PRODUCTION OF HUMAN THERAPEUTIC PROTEINS

Tamás Emri

Protein Biotechnology - Lecture 12

(159)

Insulin

Gly Ile

Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu

Asn Tyr

Cys Asn Phe

Val

Asp Glu Hys Leu Cys Gly Ser Hys

Leu Val Glu Ala Leu Tyr Leu

Val

Cys

Gly

Thr Lys Pro Thr Tyr Phe Phe Gly Arg Glu

(160)

0 2 4 6 8 10 12 14 16 18 20

-2 0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Con centrati on in the serum

aspart, lispro, glulisine insulin

NPH detemir

glargine

Pharmacokinetic properties of different insulin variants

(161)

The hepatitis B virus

(162)

Simplified life cycle of Hepatitis B virus

(163)

Geographic distribution of hepatitis B genotypes

(164)

The expression vector of Hansenula polymorpha

(165)

PRODUCTION OF HUMAN THERAPEUTIC ENZYMES

Tamás Emri

Protein Biotechnology - Lecture 13

(166)

Urate oxidase (uricase or urate oxygen oxidoreductase)

xanthine

allantoin → ureate + glyoxylate urate

5-hydroxyisourate

N H

N N H N H

O O

O

N H

N N N H

O O

O O H

₂

O + O

₂

+ H

₂

O

₂

H

-

5-hydroxyisourate

(167)

Human a-galactosidase

N-acetyl glucosamine mannose

galactose sialic acid fucose

P P

(168)

Human glucocerebrosidase

glucosylceramide

glucose ceramide

O N

O H O

H O H

O O H

n

1 2

O H O H O

H O H

O O H

O H

N

O H

O n

+H ₂O

(169)

PRODUCTION OF DIAGNOSTIC ENZYMES

Tamás Emri

Protein Biotechnology - Lecture 14

(170)

Reaction catalyzed by GOX (glucose oxidase)

b-D-glucose D-glucono-d-lactone

D-gluconic acid O

O H O

H O H O

H

O H

O

O O

H O H O

H

O H

O

H O H O

H

O H

C O O H O ₂

+ + H ₂O ₂ GOX

+H ₂O