IsolationPurificationCharacterisation of Plant Proteins VTK2010
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Transcript of IsolationPurificationCharacterisation of Plant Proteins VTK2010
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,
of Proteins from Plants
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I) Protein Isolation: Sources (expression) of plant proteins
. .
Advantages:
- Correct folding
- orrec pos - rans a ona process ng e.g. nser on o ac ve cen re, p osp ory a on,
glycosylation, cleavage, ...)
- No cloning needed
Disadvantages
- Usuall lower ield
- Often many similar proteins in the same organisms difficulties with purification
root freezing in liquid nitrogen and hydroponic plant cultivation in Kpper lab
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I) Protein Isolation: Sources (expression) of plant proteins
Heterologous expression (e.g. in bacteria, yeast or insect cells)
- Usually higher yield
- Specific expression of one protein in large amounts and possibly with specific tag
. . -
Disadvantages
- Often problems with folding, in particular in the case of large proteins and integral
membrane proteins
- Insertion of complitated cofactors (e.g. iron-sulfur clusters) is difficult or impossible,
other post-translational modifications may be missing or different compared to the
native protein possibly wrong conclusions about functions/mechanisms
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I) Protein Isolation: Methods for isolation
Used for hard tissues and cells like roots, stems, but also for hard-walled cells like
some algae and cyanobacteria
ow empera ure pro ec s e pro e ns ur ng gr n ng
Time consuming (manual grinding) or requiring suitable machinery (expensive
solution: lab mill, several thousand , cheap solution: wheat mill, about 200 )
Liquid nitrogen cooled
wheat mill in Kpper lab
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I) Protein Isolation: Methods for isolation
Used for soft tissues like some leaves, and a post-treatment after grinding
Does not require freezing and thus may avoid artefacts of freezing, but may cause
ar e ac s y ea ng o e samp e
Requires expensive (usually several thousand ) machinery
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I) Protein Isolation: Methods for isolation
Used for individual cells (plant cell culture, algae or bacteria) without or with soft walls
Does not require freezing and thus may avoid artefacts of freezingequ res very expens ve usua y many ousan mac nery
From: www.diversified-equipment.com From: www.pegasusscientific.com
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I) Protein Isolation: Methods for isolation
Used only for bacteria or animal cells
May cause degradationo mac nery nee e
From: bio-ggs.blogspot.com/2009/11/ggs-live-western...
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II) Protein Purification: Overview of Principles
Separation by expression site
Selective use of tissues or organelles
Separation by size
ra ra on Size exclusion chromatography
Preparative native gel electrophoresis
Separation by charge
Ion exchan e chromato ra h Isoelectric focussing (as chromatography, in solution or in gel electrophoresis)
Separation by specific binding sites Metal affinity chromatography using natural or artificial metal binding sites
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Magnetic separation using magnetically tagged antibodies
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II) Protein Purification: Separation by size
via size exclusion chromato ra h
Principle: Small proteins can enter more of the pores in the column material thanlarge proteins, so that small proteins migrate slower
From: elchem.kaist.ac.kr From: http://en.wikipedia.org/wiki
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II) Protein Purification: Separation by size in native gels
Principle: Small proteins are less retained by the fibers of the gel than large proteins,
so that small proteins migrate faster
foto of a green gel (Kpper et al., 2003, Funct Plant Biol) From: www.columbia.edu/.../c2005/lectures/lec6_09.html
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II) Protein Purification: Separation by charge
,
they elute at higher salt concentrations in the buffer
than less charged proteins
From: www.ucl.ac.uk/~ucbcdab/enzpur/ionX.htm
foto of phycobiliprotein purification in the lab of H. Kpper on a MonoQ anion exchange column
II) Protein Purification:From:
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II) Protein Purification:
Separation by binding sites
From:
dolly.biochem.arizona.edu/.../methods.html
foto of TcHMA4 purification in the lab of H. Kpper on an IMAC column
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III) Protein Identification: Overview of Principles
Size determination
Size exclusion chromatography or SDS PAGE . .
es ern o ng Binding to specific primary antibody, detected via labelled or enzymatically active
secondary antibody
Biochemical assays in native gels
Identification of enz mes b their characteristic activit Identification of metalloproteins by their metal content
Mass Spectrometry Fragmentation of the protein, identification of fragment sizes, and subsequent
compar son o a rary o nown ragmen a on pa erns
N-terminal Seqencing (Edman degradation)
Sequential chemical removal of individual amino acids from the N-terminus
III) P t i Id tifi ti b i ti l
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III) Protein Identification by assays in native gels Exam le of a metallo rotein identification in native els: Scannin of the els with
laser ablation inductively coupled mass spectrometry (LA-ICP-MS) for obtaining
information about metal binding to individual bands of a gel
from: Polatajko A, Azzolini M, et al (2007) JAnalAtomSpect 22, 878-887
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III) P t i Id tifi ti M S t t
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III) Protein Identification: Mass SpectrometryPrinciple
1) protein band is cut of out gel
2) protein is digested into peptides3 mass s ectrometr
4) comparison of the fragment sizes with a database
5) assignment of likely sequences to fragments
7) result: list of proteins from the database that have a similar fragmentation pattern
, , , -
III) Protein Identification: Mass Spectrometry
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III) Protein Identification: Mass Spectrometry
3) mass spectrometry
4) comparison of the fragment sizes with a database
. . . . .
III) Protein Identification: Mass Spectrometry
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III) Protein Identification: Mass Spectrometry
3a) further fragmentation of one of the fragments from the digest in the mass spectrometer, then
again mass spectrometry (MSMS)4a com arison of the fra ment sizes with a database
from: http://www.med.monash.edu.au/biochem/facilities/proteomics/maldi.html
III) Protein Identification: Mass Spectrometry
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III) Protein Identification: Mass Spectrometry
5) assignment of likely sequences to fragments
6) comparison of the fragment sequences with a database
7) result: list of proteins from the database that have a similarfragmentation pattern
from: http://www.med.monash.edu.au/biochem/facilities/proteomics/maldi.html
III) Protein Identification: Mass Spectrometry
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III) Protein Identification: Mass Spectrometry
from: Gingras AC et al, 2004, J Physiol 563, 11-21
Limitations and artefacts
contamination of gel bands with other proteins identification of the contamination(very common: keratin from skin!) instead of the protein of interest
not all proteins have the same detection efficiency (problems e.g. if many cysteines
resent even small contaminations sometimes lead to wron identifications
not all proteins are in the databases database may show results that have a similar
fragmentation pattern, but are otherwise unrelated
III) Protein Identification: N terminal sequencing
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III) Protein Identification: N-terminal sequencing
identification of the
amino acid byreversed phase HPLC
and comparison with
HPLC of standard
from: www.ufrgs.br/depbiot/blaber/section4/section4.htm
III) Protein Identification: N terminal sequencing
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III) Protein Identification: N-terminal sequencing
Sample preparation
1) run acrylamide gel (denaturating or native) -
3) stain the membrane with ponceau red (CBB and other stains also work)
4) submit for sequencing
Limitations and artefacts
problems with contaminations
in eukaryotic proteins, the N-terminus is often blocked (e.g. by methylation), which
re uired com licated de-blockin rocedures. Also non- ol merised acr lamideremains in the gel can cause blocking, so let the gel polymerise over night
Cys residues cannot be detected, and also glycosylated redidues may appear as
IV) Protein Characterisation: Overview of Principles
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IV) Protein Characterisation: Overview of Principles
Size determination
Charge determination
Analysis of the 3-dimensional Structure
Activity tests
IV) Protein Characterisation: Size determination
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) ote C a acte sat o S e dete at o
Comparison with predicted size by native and denaturating gel electrophoresis and by
size exclusion chromatography can show native oligomerisation, post-translational
modification but also artefactual degradation/aggregation
116 KDa
200 KDa SDS gel andWestern blot of
97 KDa
66KDa size shows
ost-translational
45KDa
processing as
cDNA sequence
Parameswaran
Leitenmaier et al.,
2007, BBRC)
IV) Protein Characterisation: Charge determination
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) g
protein is zero, i.e. balance between protonation of carboxyl groups and deprotonation
of amino groups is achieved
pH
neu ra reg on
acidic region
from: commons.wikimedia.org
IV) Protein Characterisation: Analysis of cofactors
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) y
Metal content (about 30% of all proteins are metalloproteins!): AAS, ICP-MS/OES,
EDX/PIXE/XRF
UV/VIS spectrum
Cadmium
bindin causesligand-metal-
charge-transfer
indicatingcysteine ligation
(Leitenmaier and
,
IV) Protein Characterisation: Analysis of cofactors
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Metal ligands: EXAFS, EPR, heteronuclear NMR provide information about ligand types
) y
and their spatial arrangement around the active site (each of these techniques has
different strenghts)
EXAFS spectrum
-
mixed S/N
li ands multi lescattering shows
histidine
Kpper, unpublished)
IV) Protein Characterisation: Analysis of the 3D Structure
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Circular Dichroism CD S ectrosco
From: www.proteinchemist.com/cd/cdspec.html
information about proportions of secondary structure types in a protein
particularly useful when X-ray crystallography and NMR are not applicable
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IV) Protein Characterisation: Activity testsTit ti f ith it b t t ( ) l bi di t t d ibl
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Titration of an enzyme with its substrate(s) reveals binding constants and possible
su s ra e n on
Cu-ATPase
Hung et al. Biochem.J.
2007, 401
Both are P1B-Type ATPases
showing the same activation
attern b their metal after
300
350
n
Cd
reconstitution into artificial
lipid vesicles
200
250
rate[1/s]
100
150
turnover
(purified by
Leitenmaier and
0,01 0,1 1 10
0
conc. metal[M]
pper
IV) Protein Characterisation: Activity tests
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Two-dimensional data, e.g. substrate concentration and temperature reveal insights
into interactions between factors that are decisive for enzyme activity, e.g.
temperature dependence of substrate binding and influence of the substrate on thethermostabilit of the enz me
mol ATP.s-1
Activity of TcHMA4
(Leitenmaier, Witt, Witzke
an pper, unpu s e
Example: Flowchart of expression, isolation, purif ication and
characterisation of the Cd/Zn ATPase TcHMA4
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characterisation of the Cd/Zn-ATPase TcHMA4
Grow plants expressing TcHMA4 hydroponically, harvest and freeze roots
Grind the frozen roots with isolation buffer in li uid N thaw
Centrifuge, discard the supernatant (soluble proteins) and resuspend the
pellet (membrane proteins) with solubilisation buffer
Centrifuge for removing insoluble residue, collect solubilised protein
Immobilized Metal Affinity Chromatography on Ni-IDA column
Identify: Quantify: In SDS Metal binding: Activity tests
Western-Blotting,Edman Sequ.
gel via luorescentdye
A I P, EXAF ,UV/VIS
catalyticalproperties)
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All slides of my lectures can be downloaded from my
wor group omepage
www.un - ons anz. e epar men o o ogy or groups pper a ,
or directl
http://www.uni-konstanz.de/FuF/Bio/kuepper/Homepage/AG_Kuepper_Homepage.html