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The Protein Purification Facility


HOME
Table of Content

Selected Protocols
EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS

Dr. Mario Lebendiker

Entries Since September 2006
mariol@cc.huji.ac.il

Tel: 972-2-6586920

mario

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.

Protein Information Submission Form  (pdf)

Table of Content

EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
HOME
Bacterial Exression Systems

GST
poly His 
Maltose
Calmodulin
Intein
Biotinylated
SUMO
Two Step: poly His and Strep-Tag
Solubility enhancement tags (SETs)
Cold Expression vector and Trigger Factor
Single Protein Production
Profinity eXact™
Corynebacterium
Pseudomonas fluorescens
ESETEC Secretion System
Brevibacillus Expression System
Cascade
Preparation of Cell Lysates /
Different Disruption Methods 

Mitochondria, Nuclear & Membrane Isolation
Purification Strategy -
and more


Test Tube
Removal of Nucleic Acids 
Buffer for Tag purification

Cleaning and Regeneration of Resins
Columns
High-throughput process development    (HTPD)
Purification of large biomolecules

Protein Quantitation 
Absorbance 280nm
BCA
Biotinylated proteins
Biuret
Bradford
CBQCA
Comparision of Methods>
Coomasie
ESL
Glycoprotein Carbohydr.
Interfering Substances
Lowry
Nano Orange
Non-Interfering
OPA(fluorescent)
Preparation Reagent
Protein Precipitation
COURSES

Publications Related to The Protein Purification Unit

Recommended Links

Recommended Publications
Drosophila Expression System

Mammalian Expression
Systems


Insect Expression System

Leishmania Expression Systems


Yeast
Expression Systems

Bryophytes (Mosses)


Choice of Buffers
Purification of Recombinant Proteins
GST
poly His 
Maltose 
Calmodulin
Intein 
T7.Tag
Cellul.Bind.Domain
Nus A
Biotinylated 
SUMO
StrepTag
FLAG
Halo tag Fusion
Solubility enhancement tags (SETs)
Fluorescent Protein
Cold Expression
LYTAG Two PhaseSystem
Profinity eXact™
Bispecific Affinity
CaptureSelect C‐tag
Gel Electrophoresis

PAGE-SDS (Laemmli)
Basic-Native
Acidic-Native
Tricine
Phosphate affinity SDS-PAGE
QPNC-PAGE

Protein extraction from Polyacrylamide Gel

Proteomics Sample Preparation
Protein Precipitation

Protein extraction from Polyacrylamide Gel 

Western-Blot

Gel Stain

Coomasie Blue
Fluorescence
Glycoprotein
Lypopolysaccharide

Oligohistidine
Phosphoprotein
Silver Stain
Zinc Stain
In-Vitro Translation
Inhibitors

Apoptosis Inhibitors
Cell Division / Cell Cycle / Cell Adhesion
Inhibitors
Lipid Signal
Inhibitors
Neurobiology/
Neurodegeneration Inhibitors
Nitric Oxid/Oxidative Stress
Inhibitors
Phosphorilation / Dephosphorilation
Protease Inhibitors

Others

Cleavage of Recombinant Proteins

Cleavage Sites Table for Fusion Proteins

Factor-Xa
Thrombin
Enterokinase
PreScission
TAGZyme
TEV-Protease
HRV 3C
SUMO

Proteases



DNA removal


Endotoxin removal


Cell Penetration
Affinity Chromatography
Activated resins
Antibody Purification and Antibody fragmentation
Lectins and Glycoprotein
Immunoprecipitation
Mimetics Ligands, Antibody fragments and Similar Technology
General

LINKS
A detailed collection of information on initial bioinformatic investigation
using bioinformatic tools to strategically design expression/purification projects, can be found on the website page of  Dr. Nurit Kleinberger-Doron inside the website of the  Protein Expression Facility directed by Dr Tsafi Danieli (Wolfson Centre for Applied Structural Biology - Hebrew University of Jerusalem).


Chromatofusing

Expanded Bed Adsorption

Gel Filtration

Hydrophobic Interaction

Hydroxyapatite

Ion Exchange

Multimodal and Hydrophobic Charge-Induction Chromatography 

Reverse Phase

Thiophilic


Protein-Protein Interaction

Analytical 
Ultracentrifugation

Light Scattering

Circular Dichroism


High Abundant Serum Proteins Removal

Crystallography and Recombinant Methods

Phospho-Protein Purification

Polyubiquitin-modified proteins

Protein Aggregation

Protein Labeling with Biotin

Protein Refolding / Inclusion Bodies

Simulated Moving Bed Chromatography

Storage of Purified Proteins

Viral Purification


Selected Protocols of The Protein Purification Unit and Others

Preparation of Cell Lysates / Different Disruption Methods
Bacterial Protein Extraction (mini-scale) Sonication

Bacterial Protein Screen (DDM lysis) - Small affinity binding
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 - Endotoxin removal
Removal of Nucleic Acids - Different Protocols
Endotoxin Clearance with Triton X-114 – Selected protocols from the literature

Purification Strategy - Others
Test Tube
Buffer solubility screen to avoid protein aggregation during concentration

Limited proteolysis - Partial Proteolytic Digestion

Purification of Recombinant Proteins
Small scale GST-fusion protein purification under nature conditions

Small and large scale His-Tag fusion protein purification under nature conditions

Small scale His-Tag fusion protein purification under denaturative conditions

Small and large scale MBP-fusion protein purification 
Protein Refolding on IMAC resin - Batch Screening Procedure - On-Column Scale-up
Additives Used to Increase Folding and Prevent Aggregation
Contaminant Removal from Inclusion Bodies Before Solubilization

Cleavage of Recombinant Proteins

Cleavage Sites Table for Fusion Proteins
Factor Xa Cleavage of a MBP fusion protein
Thrombin Cleavage of a GST fusion protein

Protein Refolding

Protein Refolding on IMAC resin - Batch Screening Procedure - On-Column Scale-up
Additives Used to Increase Folding and Prevent Aggregation
Contaminant Removal from Inclusion Bodies Before Solubilization

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)

Amsili S, Zer H, Hinderlich S, Krause S, Becker-Cohen M, MacArthur D, North K and Mitrani-Rosenbaum S (2008) 
UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) Binds to Alpha-Actinin 1: Novel Pathways in
Skeletal Muscle?
PLoS ONE 3(6): e2477 1-9
.  (pdf)

Steiner-Mordoch S, Soskine M, Solomon D, Rotem D, Gold A, Yechieli M, Adam Y and Schuldiner S (2008)
Parallel topology of genetically fused EmrE Homodimers
The EMBO Journal 27, 17–26   (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)

Yannay-Cohen N.,Carmi-Levy I.,Kay G.,Yang C.,Min HanJ. ,Kemeny M., Kim S.,Nechushtan H.,and Razin E.  (2009)
LysRS Serves as a Key Signaling Molecule in the Immune Response by Regulating Gene Expression
Molecular Cell 34, 603–611   (pdf)

Tabib A., Krispin A., Trahtemberg U., Verbovetsk I., Lebendiker M., Danieli T., and Mevorach D. (2009)
Thrombospondin-1-N-terminal domain (heparin binding N-terminal domain) induces a phagocytic state, and Thrombospondin-1-C-terminal
domain induces a tolerizing phenotype in dendritic cells.

PLoS ONE 4(8) e6840 1-7   (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)

Kasherman Y,Sturup S,and Gibson D  (2009)
Is Glutathione the Major Cellular Target of Cisplatin? A Study of the Interactions of Cisplatin with Cancer Cell Extracts
J. Med. Chem., 52, 4319–4328   (pdf)

Elbaz Y, Danieli T, Kanner B and Schuldiner S (2010) 
Expression of neurotransmitter transporters for structural and biochemical studies.

Protein Expr Purif.  73:152-160.   (pdf)


Lebendiker M., and Danieli T. (2011)
Purification of Proteins fused to Maltose-binding Protein.
Protein Chromatography: Methods and Protocols. Editor(s): Dermot Walls, Sinéad T. T. Loughran Series:
Methods in Molecular Biology Vol.681: 281-293  
(pdf)

Tzarum N., Diskin R., Engelberg D. and Livnah O.(2011)
Active Mutants of the TCR-Mediated p38α Alternative Activation Site Show Changes in the Phosphorylation Lip and DEF
Site Formation
J. Mol. Biol. 405, 1154–1169  (pdf)    

Reingewertz T., Shalev D., Sukenik S., Blatt O., Rotem S., Lebendiker M., Larisch S. & Friedler A. (2011)
Mechanism of the Interaction between the Intrinsically Disordered C-terminus of the Proapoptotic ARTS Protein and the Bir3
Domain of XIAP 

PLoS ONE  vol 6, issue 9, e24655   (pdf)

Siman P., Blatt O., Moyal T., Danieli T.,Lebendiker M., Lashuel H., Friedler A. and Brik A. (2011)
Chemical Synthesis and Expression of the HIV-1 Rev Protein                                                             
ChemBioChem 12 (7): 1097-1104 - DOI: 10.1002/cbic.201100033  (pdf)

Weiss S., Kohn E., Dadon D., Katz B., Lebendiker M., Kosloff M., Colley N. and Minke B. (2012)
Ca2+segregation mediated by calphotin prevents light induced retinal degeneration.
The Journal of Neuroscience 32(42):14696 –14708   (pdf)

Gabizon R, Brandt T, Sukenik S, Lahav N, Lebendiker M, Shalev D, Veprintsev D and Friedler A. (2012)  
Modulating the oligomerization equilibrium of p53 by peptides that bind its C terminal domain.
PLoS ONE 7 (5)
e38060 - doi:10.1371/journal.pone.0038060   (pdf)  

Amartely H , David A, Lebendiker M, Benyamini H, Izraeli S and Friedler A (2013)
The STIL protein contains intrinsically disordered regions that mediate its protein-protein interactions
Chemical Communication DOI: 10.1039/c3cc45096a  (pdf)

Lebendiker M., and Danieli T. (2014)
Production of prone-to-aggregate proteins (Review)
FEBS Letters 588: 236–246
- DOI information: 10.1016/j.febslet.2013.10.044  (pdf)

Buch M., Wine Y., Dror Y., Rosenheck S., Lebendiker M., Giordano R., Leal R., Popov A., Freeman A and Frolow F. (2014)
Protein Products Obtained by Site-Preferred Partial Crosslinking in Protein Crystals and ‘‘Liberated’’ by Redissolution.
Biotechnology and Bioengineering DOI 10.1002/bit.25186  
(pdf)

Joubran S., Zigler M, Pessah N., Klein S., Shir A., Edinger N.Sagalov A., Razvag Y., Reches M., and Levitzki A. (2014)  
Optimization of Liganded PolyethyleniminPolyethylene Glycol Vector for Nucleic Acid Delivery

dx.doi.org/10.1021/bc500252a  Bioconjugate Chem.  
(pdf)

Sharabi O., Shirian J., Grossman M., Lebendiker M., Sagi I, and Shifman J.  (2014)
Affinity- and specificity-enhancing mutations are frequent in multispecific interactions between TIMP2 and MMPs                                                                                          
PLoS ONE 9(4): e93712. doi:10.1371/journal.pone.0093712  (pdf)

Lebendiker M., Danieli T. and de Marco A. (2014)                                                      
The Trip Adviser guide to the protein science world: a proposal to improve the awareness concerning the quality of recombinant proteins.                                     
BMC Research Notes (2014) 7:585  doi:10.1186/1756-0500-7-58  
(pdf)  (pdf-figure)

Maes M.; Amit E.; Danieli T.; Lebendiker M.; Loyter A.and Friedler A. (2014)         
The disordered region of Arabidopsis VIP1 binds the Agrobacterium VirE2 protein outside its DNA binding site                                                                                
Protein Engineering, Design, and Selection" (PEDS)1-8 doi:10.1093/protein/gzu036  
(pdf)

Buch M., 

Weiss S.

Recommended Links 

Protein Expression Facility - Dr Tsafi Danieli - Wolfson Centre for Applied Structural Biology - Hebrew University of Jerusalem
A detailed collection of information on expression system and cloning strategies

Initial Bioinformatic Investigation - Dr Nurit Kleinberger Doron - Hebrew University of Jerusalem
A detailed collection of information on initial bioinformatic investigation, and the use of  bioinformatic tools to strategically design expression/purification
projects

ExPASy - ProtParam: A tool which allows the computation of various physical and chemical parameters for a given protein. The computed
parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life,
instability index, aliphatic index and grand average of hydropathicity (GRAVY)

PROTEIN CALCULATOR v3.3 - Scripps : A tool which allows the computation of various physical and chemical parameters for a given
protein. The computed parameters include the molecular weight, theoretical pI, amino acid composition, atomic composition, extinction
coefficient, charge, protease cleavage.

Recombinant Protein Solubility Prediction - University of Oklahoma:  
The statistical model predicts protein solubility assuming the protein is being overexpressed in Escherichia coli. Reference: Predicting the solubility
of recombinant proteins in Escherichia coli. Wilkinson et al. Biotechnology (N Y). 1991 May;9(5):443-8.

ESPRESSO (EStimation of PRotein Expression and SOlubility):
A sequence-based predictor for estimating protein expression and solubility

ExPASy - Peptide Cutter:  
Predicts potential protease and cleavage sites and sites cleaved by chemicals in a given protein sequence.

FoldIndex© - Weizmann Institute of Science: 
FoldIndex©: a simple tool to predict whether a given protein sequence is intrinsically unfolded. Prilusky et al.
BIOINFORMATICS APPLICATIONS
NOTE Vol. 21 no. 16 2005, pages 3435–3438 doi:10.1093/bioinformatics/bti537

CYSPRED - Predictor of the bonding state of cysteines in proteins     
The role of evolutionary information in predicting the disulfide bonding state of cysteines in proteins- Fariselli et al.  Proteins 36: 340-346 (1999)

DISULFIND - Cysteines Disulfide Bonding State and Connectivity Predictor - Univ. Of Firenze    
A server for the prediction of the bonding state and connectivity pattern of the cysteines in a given amino acid sequence

DiANNA 1.1 - Cysteine state and Disulfide Bond partner prediction - Boston College
Determines the cysteine species: free cysteine, half-cystine or ligand-bound; and if a cysteine is predicted to be ligand-bound, then the most likely of
the four most common ligands (iron, zinc, cadmium, carbon)

GlobPlot - Intrinsic Protein Disorder, Domain & Globularity Prediction - EMBL   GlobPlot: exploring protein sequences for globularity and
disorder.  Linding et. al. Nucleic Acids Research, 2003, Vol. 31, No. 13 3701–3708        

Stormoff:  Weight to Molar Quantity (for proteins)
This program helps you to convert the weight (weight concentration) in the molar quantity (molar concentration) 
and vice versa.

Ammonium Sulfate Calculator - EnCor Biotechnology Inc.
The program calculates how much solid Ammonium Sulfate you need to add to a specific volume of a solution to get a specific percentage
saturation at a specific temperature.

CRAPome: a contaminant repository for affinity purification–mass spectrometry data  (pdf)  
A web-accessible resource that stores and annotates negative controls generated by the proteomics research community and enables their use for
scoring AP-MS data.


Recommended Publications

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)
Geerlof A. et.al.  The impact of protein characterization in structural proteomics.
    Acta Cryst. (2006) D62: 1125-1136   (pdf) 
Newby Z. et.al.  A general protocol for the crystallization of membrane proteins for X-ray structural investigation.
    Nature Protocols (2009) 4: 619-637  (pdf)  
Lee J. et.al.  An efficient platform for screening expression and crystallization of glycoproteins produced in human cells.  
    Nature Protocols (2009) 4: 592-604  (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).
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Dr. Mario Lebendiker The Protein Purification Facility
mariol@cc.huji.ac.il Tel: 972-2-6586920

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