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Faculty of Science |
The Protein Purification Facility
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Table of Content Selected Protocols |
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Tel: 972-2-6586920
The Protein Purification Facility is a resource of information and assistance available to researches and students as well as biotech and pharmaceutical companies that are interested in protein purification. Our Unit assists researches to overcome the major bottleneck in structure determination by X-Ray crystallography or NMR, that is the preparation of suitable crystalline samples. Mainly we provide consultation and active support for researchers and students who are interested in using the equipment, methods and materials of the Facility.
The unit offers a complete and fully automated liquid chromatography system designed for method development and research applications, that simplifies the transition from laboratory to full-scale production. We have columns and resins for purification according to size, charge, hydrophobicity and substrate affinity. Gel electrophoresis and IEF (isoelectric focusing) apparatus; cell disruption, blotting and ultrafiltration systems.
The facility assists researchers, students and people from the biotechnology industry in resolving their protein purification problems. We are actively involved in many collaborations for structural and biochemical studies; isolation and identification of new proteins.
Due to the unique equipment and wide experience in the field, the protein purification facility is the only place in Israel that provides comprehensive services in this area, and also functions as a learning station.
Preparation
of Cell Lysates / Different Disruption Methods
Bacterial
Protein Extraction (mini-scale) Sonication
Bacterial
Protein Extraction (mini-scale) Using B-Per
PROTEIN EXPRESSION
FACILITY of THE HEBREW UNIVERSITY Sonication
of bacterial samples
Choice of Buffers
AMERSHAM
BIOSCIENCES Recommended buffers for Anion Exchange Chromatography
AMERSHAM BIOSCIENCES
Recommended buffers for Cation Exchange Chromatography
AMERSHAM
BIOSCIENCES Recommended Volatile buffers systems for Ion Exchange
Chromatography
PROTEIN
EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR
BIOLOGY LABORATORY Choice
of lysis buffer and additives
PROTEIN
EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR
BIOLOGY LABORATORY Solubility
Studies Preparation
of soluble/insoluble protein from cells
DNA removal
Removal of Nucleic Acids - Different Protocols
Purification Strategy
- Others
Test Tube
Purification of Recombinant Proteins
Small scale GST-fusion
protein purification under nature conditions
Small
scale His-Tag fusion protein purification under nature conditions
Small scale His-Tag
fusion protein purification under denaturative conditions
Small
scale MBP-fusion protein purification
Protein Refolding
Additives Used to Increase Folding
and Prevent Aggregation
PROTEIN
EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR
BIOLOGY LABORATORY In
vitro denaturation and refolding
Contaminant
Removal from Inclusion Bodies Before Solubilization
Storage of Purified Proteins
PROTEIN
EXPRESSION AND PURIFICATION FACILITY OF THE EUROPEAN MOLECULAR
BIOLOGY LABORATORY Storage
of Purified Proteins
Protein
Quantitation
Comparision
of Different Protein Determination Methods
Absorbance 280nmAccording
to Protein Protocols in CD Rom
Bradford
Lowry According
to Protein Protocols in CD Rom
Lowry-Peterson
For membrane proteins, diluted solutions or solutions with
s interferant
Protein
Precipitation Methods to concentrate or eliminate interferences
before electrophoresis or protein determination
Gel Electrophoresis
PAGE-SDS (Laemmli)
Basic-Native:
For Acidic and Neutral proteins (pI < 7.0)
Acidic-Native:
For Basic proteins (pI > 7.0)
Tricine:
SDS gel for low MW proteins (<25 kDa)
Protein
Precipitation Methods to concentrate or eliminate interferences
before electrophoresis or protein determination
Silver
Stain
Publications Related to The Protein Purification
Unit
Schwaiger, M., Lebendiker, M., Yerushalmi,H.,
Coles, M., Groger, A., Schwartz, C., Schuldiner,S.,and Kessler,
H. (1998).
NMR-Investigation of the Multidrug Transporter Emr-E
an Integral Membrane Protein.
Eur. J. of Biochemistry
254: 610-619. (pdf)
Muth T.R. and Schuldiner S. (2000)
A membrane-embedded
glutamate is required for ligand binding to the multidrug transporter
EmrE.
EMBO J. 19: 234-240 (pdf)
Tate C., Kunji E., Lebendiker M., and Schuldiner
S. (2001).
The projection structure of EmrE, a proton linked multidrug
transporter from Escherichia coli, at 7Å resolution.
The
EMBO Journal 20: 77-81 (pdf)
Orzech E., Okhrimenko H., Reich V., Cohen S., Weiss
A., Melamed-Book N., Lebendiker M., Altschuler Y.and Aroeti
B.(2001).
The AP-1 adaptor of the clathrin coat associates with
microtubules via microtubule associated proteins.
J.of Biol.
Chemistry 276 (33): 31340-31348 (pdf)
Ben-Zimra M, Koler M, and Orly J. (2002)
Transcription
of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta:
Activating Protein-2 Assumes the Role of Steroidogenic Factor-1
by Binding to an Overlapping Promoter Element.
Mol. Endocrinol.16:
1864-1880 (pdf)
Fish A, Lebendiker M, Nechushtai R & Livnah
O (2003).
Purification, crystallization and preliminary X-ray analysis
of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus
laminosus.
Acta Crys. D-59: 734-736 (pdf)
Zhang M, Fishman Y, Sher D and Zlotkin E (2003).
Hydralisin,
a Novel Animal Group-Selective Paralytic and Cytolytic Protein
from Noncnidocystic Origin in Hydra.
Biochemistry 42:
8939-8944 (pdf)
Rosen G, Nisimov I, Helcer M, and Sela M (2003)
Actinobacillus
actinomycetemcomitans Serotype b Lipopolysaccharide Mediates Coaggregation
with Fusobacterium nucleatum.
Infection and Immunity 71 (6):
3652–3656 (pdf)
Listovsky T, Oren Y, Yudkovsky Y, Mahbubani H, Weiss
A, Lebendiker M and Brandeis M (2004)
Cdh1/Fzr mediates
its own degradation
The EMBO Journal 00: 1–8 (pdf)
Soskine M, Adam Y, and Schuldiner S (2004)
Direct Evidence for Substrate-induced
Proton Release in Detergent-solubilized EmrE, a Multidrug
Transporter
J.of Biol. Chemistry 279 (11): 9951-9955
(pdf)
Neubig R, and Roman D, (2004)
Sites of interest on the World Wide Web
Mol.
Interv.4: 298 (pdf)
Klein S, Geiger T, Linchevski I, Lebendiker M, Itkin A, Assayag
K and Levitzki A (2005)
Expression and purification of active
PKB kinase from Escherichia coli.
Protein Expression and Purification
41: 162–169 (pdf)
Sher D., Fishman Y., Zhang M., Lebendiker M., Gaathon A.,
Manchenio J. and Zlotkin E. (2005)
Hydralisins: a new category
of diverse Beta-Poreforming toxins in Cnidaria. Characterization
and preliminary structure-function analysis.
J. of Biol. Chemistry
280: 22847 - 22855 (pdf)
Fish A., Danieli T., Ohad I., Nechushtai R. and Livnah O. (2005)
Structural
Basis for the Thermostability of Ferredoxin from the Cyanobacterium
Mastigocladus laminosus.
J. Mol. Biol. 350: 599–608 (pdf)
Copty A., Sakran F., Popov O., Ziblat R., Danieli T., Golosovsky M.and
Davidov D. (2005)
Probing of the microwave radiation effect
on the green fluorescent protein luminescence in solution
Synthetic
Metals 155: 422–425 (pdf)
Elia N., Frechter S., Gedi Y., Minke B., and Selinger Z. (2005)
Excess
of Gße over Gqαe in vivo prevents dark, spontaneous activity of
Drosophila photoreceptors.
The Journal of Cell Biology 171(3):
517-526 (pdf)
Soskine M., Mark S., Tayer N., Mizrachi R. and Schuldiner S. (2006)
On
Parallel and Antiparallel Topology of a Homodimeric Multidrug Transporter
J. of Biol. Chemistry 281: 36205–36212 (pdf)
Taylor A. , Haze-Filderman A, Blumenfeld A., Shay B., Dafni L., Rosenfeld
E., Leiser Y., Fermon E., Gruenbaum-Cohen Y.and Deutsch D. (2006)
High yield of biologically active recombinant human amelogenin
using the baculovirus expression system.
Protein Expression
and Purification 45: 43–53 (pdf)
Diskin R., Lebendiker M., Engelberg L. And Livnah O. (2007)
Structures
of p38α Active Mutants Reveal Conformational Changes in L16 Loop
that Induce Autophosphorylation and Activation.
J. Mol. Biol.
365, 66–76 (pdf)
Funkenstein B. and Rebhan Y. (2007)
Expression, purification, renaturation
and activation of fish myostatin expressed in Escherichia coli: Facilitation
of refolding and activity inhibition by myostatin prodomain.
Protein
Expression and Purification 54: 54–65 (pdf)
Hayouka Z., Rosenbluh J., Levin A., Loya S., Lebendiker M., Veprintsev
D., Kotler M., Hizi A., Loyter A. & Friedler A. (2007)
Inhibiting
HIV-1 Integrase by Shifting its Oligomerization Equilibrium
PNAS
104 (20): 8316-8321 (pdf)
Weiss Y., Bromberg Z., Raj N., Raphael J., Goloubinoff P., Ben-Neriah
Y.and Deutschman D. (2007)
Enhanced heat shock protein 70 expression alters
proteasomal degradation of I[kappa]B kinase in experimental acute respiratory
distress syndrome.
Critical Care Medicine. 35(9):2128-2138 (pdf)
Coster G, Hayouka Z, Argaman L, Strauss C, Friedler A, Brandeis M and
Goldberg M (2007)
The DNA Damage Response Mediator MDC1 Directly Interacts with the Anaphase-promoting Complex/Cyclosome
J. of Biol.
Chemistry 282 (44): 32053–32064 (pdf)
Shalev-Malul G., Viner-Mozzini Y., Sukenik A., Gaathon A., Lebendiker
M. and Kaplan A. (2008)
An AbrB-like protein might be involved
in the regulation of cylindrospermopsin production by Aphanizomenon ovalisporum.
Environmental Microbiology 10(4), 988–999 (pdf)
Rotem S, Katz C, Benyamini H, Lebendiker M, Veprintsev D, Rudiger
S, Danieli T and Friedler A. (2008)
The structure and interactions of the
proline-rich domain of ASPP2 (2008)
J. of Biol. Chemistry 283 (27),
18990–18999 (pdf)
Mayshar Y, Rom E, Chumakov I, Kronman A, Yayon A and Benvenisty N (2008)
FGF4 And its Novel Splice Isoform Have Opposing Effects on the
Maintenance of Human Embryonic Stem Cell Self Renewal
Stem Cells
Express, published online January 10, 2008; doi:10.1634/stemcells.2007-1037
(pdf)
Katz C., Benyamini H.,Rotem S., Lebendiker M., Danieli T., Iosub
A., Refaely H., Dines M., Bronner V., Bravman T., Shalev D., Rüdiger S.,
and Friedler A. (2008)
Molecular basis of the interaction between the antiapoptotic
Bcl-2 family proteins and the proapoptotic protein ASPP2
PNAS 105
(34): 12277–12282 (pdf)
Reingewertz T.; Benyamini H.; Lebendiker M.; Shalev D.and Friedler
A.(2009)
The C-Terminal Domain of the HIV-1 Vif Protein is Natively Unfolded
in its Unbound State.
PEDS (Protein Engineering, Design, and Selection)
1–7, doi: 10.1093/protein/gzp004 (pdf)
Lieman-Hurwitz J, Haimovich M, Shalev-Malul G, Ishii A, HiharaY, Gaathon
A, Lebendiker M and Kaplan A. (2009)
A cyanobacterial AbrB-like protein
affects the apparent photosynthetic affinity for CO2 by modulating low-CO2-induced
gene expression.
Environmental Microbiology 11(4), 927–936
(pdf)
Lederman L. (2009)
Protein Isolation and Purification – Tech News
BioTechniques
46:87-89 (pdf)
Sela M., Babitski E., Steinberg D., Kohavi D. and Rosen G. (2009)
Degradation of collagen-guided tissue regeneration membranes by
proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents
Clin. Oral Impl. Res. 20, 496–502 (pdf)
Shay B, Gruenbaum-Cohen Y, Tucker AS,
Taylor AL, Rosenfeld E, Haze A, Dafni L, Leiser Y, Fermon E,
Danieli T, Blumenfeld A, Deutsch D. (2009)
High yield
expression of biologically active recombinant full length human tuftelin protein
in baculovirus-infected insect cells.
Protein Expr
Purif. 2009 Nov; 68(1):90-98 (pdf)
Crystallography and
Recombinant Proteins
Derewenda Z., The use of recombinant methods
and molecular engineering in protein crystallization. Methods
(2004), 34:354–363 (pdf)
Smyth D., REVIEW Crystal structures of fusion
proteins with large-affinity tags.Protein Science (2003),12:1313–1322
(pdf)
Production and Purification of Recombinant Proteins
Structural Genomics Consortium Protein
production and purification. Nature Methods 2008, 5: 135-146
(pdf-I) (pdf-II)
Jonasson P.,Genetic Design for Facilitated
Production and Recovery of Recombinant Proteins in E.Coli.
Biotechnol.Appl.Biochem 2002,35: 91-105 (pdf)
Nilsson J.,Affinity Fusion Strategies
for Detection, Purification and Immobilization of Recombinant
Proteins.Protein Expr. and Purif. 1997, 11: 1-16
(pdf)
Stevens R., Design of Hgh-Throughput Methods
of Protein Production for Structural Biology. Structure 2000,8:
R177-R185 (pdf)
Swartz J. Advances in E.Coli Production of
Therapeutic Proteins. Current Opinion in Biotechnology
2001,12: 195-210 (pdf)
Protein Refolding
Altamirano M., Refolding
Chromatography with Immobilized Mini-Chaperones. PNAS 1997,94:
3576-3578 (pdf)
Armstrong N.,A New Protein Folding Screen...etc.Protein
Science 1999, 8: 1475-1483 (pdf)
Chen G., Overexpression of a Glutamate Receptor
(GluR2) ligand binding domain in E.Coli: Application of a novel
protein folding screen (pdf)
De Bernardez Clark E., Refolding of Recombinant
Proteins . Current Opinion in Biotechnology 1998, 9:
157–163 (pdf)
De Bernardez Clark E., Protein refolding for
industrial processes. Current Opinion in Biotechnology
2001, 12: 202–207 (pdf).
Eiler S., Overexpression, Purification, and
Crystal Structure of Native ER alphaLBD Protein Expression
and Purification (2001) 22, 165–173 (pdf)
Machida S., Cycloamylose as an efficient
artificial chaperone for protein refolding. FEBS Letters486
(2000) 131-135 (pdf)
Middelberg A.,Preparative Protein Folding.
TRENDS in Biotechnology 2002,20 (10):
437-443
(pdf)
Ming Li et al.,In vitro protein refolding
by chromatographic procedures. Protein Expr.
and Purif. 2004,33: 1-10 (pdf)
Tsumoto K.,Practical Considerations in Refolding
Proteins from Inclusion Bodies. Protein Expr. and Purif.
2003,28: 1-8 (pdf)
Dr. Mario Lebendiker
The Protein Purification Facility
mariol@cc.huji.ac.il
Tel: 972-2-6586920
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