Additives_Folding The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mario.l@mail.huji.ac.il  Tel: 972-2-6586920 

        Protein Production for Biophysical and Biochemical Studies - PPBBS (#92632) - 2015
More than a simple His purification and PAGE-SDS analysis is necessary to prepare a protein sample for biophysical, biochemical or clinical studies.

Covered topics:

Course will be open for students and under-graduate students (Master, PhD and post-doc) that need highly purified proteins for their studies. Rules for the course
First semester (October 2015 - January 2016) every Wednesday between 9:00 to 10:45  . Room 504 - Life Science Institute.




21/10/2015

Introduction to Protein Purification. Speaker: Dr. Mario Lebendiker (HUJI)  (Literature)

Input for Purification Protocol Development - Guidelines for Protein Purification - Sample Preparation before Chromatography - Chromatography: General Considerations Speaker: Dr. Mario Lebendiker (HUJI) (Literature)

28/10/2015

Input for Purification Protocol Development - Guidelines for Protein Purification - Sample Preparation before Chromatography - Chromatography: General Considerations Speaker: Dr. Mario Lebendiker (HUJI) (Literature)

4 & 11 /11/2015

Basic  principles on protein  expression. Advantages and  disadvantages of different expression systems   (second talk)
Course92632_2014/Talks 2015/BacterialExpression2015.pdf  Speaker: Dr. Tsafi Danieli (HUJI)

18/11/2015

Ion Exchange: Main stages in  chromatography. Basis for selectivity. Operational considerations.  Determination of start conditions. Parameters for optimization. Examples.
Speaker: Dr. Mario Lebendiker (HUJI) (Literature) 

25/11/2015

Hydrophobic Exchange: Chromatography: Main stages in chromatography. Basis for selectivity. Determination of start conditions.
Operational considerations. Parameters for optimization. Examples  Speaker: Dr. Mario Lebendiker (HUJI) (Literature)          

Other Purification Techniques: Hydroxyapatite, Reverse Phase, Multimode Resins (HIC + IEX & GF + IEX)
Speaker: Dr. Mario Lebendiker (HUJI)  (Literature)     
2/12/2015 Gel Filtration Chromatography and desalting: Main stages in chromatography. Basis for selectivity. Operational considerations. Determination of start conditions. Parameters for optimization. Examples. SEC-MALS Speaker: Hadar Amartely (HUJI) (Literature)     9 and 16/12/2015 Affinity chromatography: Type of Affinities. Main stages. Advantages and Disadvantages.Genetically engineered Tags, recombinant proteins. Cleavage Sites. Parameters for optimization. Examples. (Literature) Speaker: Dr. Mario Lebendiker (HUJI) 23/12/2015 Purification Strategy: Selection and combination of purification techniques. Examples. Protein Characterization. Storage. Speaker: Dr. Mario Lebendiker (HUJI) 30/12/2015 Aggregation: How to solve aggregation problems. Mechanism of aggregation. Methods for screening solubility. Considerations when selecting buffer components.   Additives that increase yield and prevent aggregation and/or stabilize proteins.  Speaker: Dr. Mario Lebendiker (HUJI) (Literature)     6/01/2015 Quality control: Major requirements for purification of proteins for structural studies (crystallography, NMR, others) Speaker: Dr. Mario Lebendiker (HUJI) (Literature) 13/01/2015 Refolding: Major steps. Protein refolding methods. Screening. Strategies and examples.
Speaker: Dr. Mario Lebendiker (HUJI) (Literature) Membrane Proteins: The challenge of Membrane protein purification. Main features, use and removal of detergents. Practical aspects of over-expressing bacterial membrane proteins for structural studies. Detergents in crystallography
Speaker: Dr. Mario Lebendiker (HUJI)
(Literature)

Protein Production for Biophysical and Biochemical Studies -PPBBS (#92632) - 2014
                                                                    Practical Course (#92638)
More than a simple His purification and PAGE-SDS analysis is necessary to prepare a protein sample for biophysical, biochemical or clinical studies.

The Protein Purification Facility of the Hebrew University is organizing a course that comprises basic knowledge in:

Ø  FPLC system. FPLC software Ø  Classical chromatography: IEX, SEC, HIC, Affinity and Mixed Mode. Main stages. Basis for selectivity. Operational considerations. Start conditions. Parameters for optimizationØ  Genetically engineered Tags. Cleavage SitesØ  Additional techniques use in protein productionØ  Protein purification strategy and protocol development. Connection between expression and protein quality. Selection and combination of purification techniques Ø  Major requirements for purification of proteins for structural studies and others. Ø  Protein quality and characterization: SPR, ITC, MST, AUC, SEC-MALS, etc Ø  How to solve aggregation problems. Refolding. The challenge of membrane protein purification, etc

Course will be open for 40 students and under-graduate students (Master, PhD and post-doc) that need highly purified proteins for their studies. Students from all Israel universities are invited to participate.

Four days course (Two credit points): 6/11 20/11 4/12 & 18/12/2014 from 10:00 to 18:00.
10:00 – 10:50      10’ interval
11:00 – 11:50      70’ interval
13:00 – 13:50      10’ interval
14:00 – 14:50      40’ interval
15:30 – 16:20      10’ interval
16:30 – 17:30      end
 

Optional Practical Course (#92638): at the end of the Course #92632 students are invited to perform a practical short course in our Facility working in their own project
    

1st day (06/11/2014)

1)     10:00 – 10:50   Introduction to Protein Purification. Main stages. Speaker: Dr. Mario Lebendiker (HUJI)  (Literature)

2)      11:00 – 11:50   FPLC system. FPLC software  Speaker: Dr. Mario Lebendiker (HUJI) (Literature)3)      13:00 – 13:50    Ion Exchange: Main stages in chromatography. Basis for selectivity. Operational considerations. Determination of
                                   start conditions. Parameters for optimization. Speaker: Dr. Mario Lebendiker (HUJI) (Literature) 4)      14:00 – 14:50   Ion Exchange(continue), examples Speaker: Dr. Mario Lebendiker (HUJI)5)      15:30 – 16:20   Hydrophobic Exchange: Chromatography and desalting: Main stages in chromatography. Basis for selectivity.
                                   Determination of start conditions. Operational considerations. Parameters for optimization. Examples.
                                   Speaker: Dr. Mario Lebendiker (HUJI) (Literature)         

6)      16:30 – 17:30   Protein concentration: native methods, denaturative methods. Protein determination: different methods.    
                                   Speaker: Ofrah Blatt (HUJI)    

 2nd day (20/11/2014)

7)      10:00 – 10:50    Gel Filtration Chromatography and desalting: Main stages in chromatography. Basis for selectivity.
                                  Operational considerations. Determination of start conditions. Parameters for optimization. Examples.
                                  Speaker: Dr. Mario Lebendiker (HUJI)
 (Literature)                                        

8)      11:00 – 11:50    Hydrophobic Exchange: Chromatography and desalting: Main stages in chromatography. Basis for selectivity.
                                   Determination of start conditions. Operational considerations. Parameters for optimization. Examples.
                                   Speaker: Dr. Mario Lebendiker (HUJI) (Literature)         
                                   Other Purification Techniques: Hydroxyapatite, Reverse Phase, Multimode Resins (HIC + IEX & GF + IEX)
                                   Speaker: Dr. Mario Lebendiker (HUJI)  (Literature)     9)      13:00 – 13:50    Affinity chromatography: An historical introduction. Speaker: Dr. Meir Wilchek (WIS)

10)     14:00 – 14:50    Affinity chromatography: Main stages. Advantages and Disadvantages. Type of Affinities.
                                   Designing and preparing an affinity gel. Speaker: Dr. Mario Lebendiker (HUJI) (Literature)11)     15:30 – 16:20    Affinity chromatography: Genetically engineered Tags, recombinant proteins. Cleavage Sites.
                                   Parameters for optimization. Examples. Speaker: Dr. Mario Lebendiker (HUJI) (Literature)

12)     16:30 – 17:30   Dialysis. Ultrafiltration. Electrophoresis: native and denaturative gels; sample buffer
                                (+/- reducing agents or heat); stain (sensitivity); western. SEC-MALS
                               Speaker:  Hadar Amartelly (HUJI)
                    

 

3rd day (04/12/2014)

13)   10:00 – 10:50    Cell disruption considerations. Centrifugation. Filtration.  Columns, membranes.
                                   Speaker: Dr. Mario Lebendiker (HUJI)

14)   11:00 – 11:50    Input for Purification Protocol Development. Guidelines for Protein Purification.
                                    Speaker: Dr. Mario Lebendiker (HUJI) 15)   13:00 – 13:50    Selection and combination of purification techniques. Storage. Speaker: Dr. Mario Lebendiker (HUJI) 16)   14:00 – 14:50    How to solve aggregation problems. Mechanism of aggregation. Methods for screening solubility.
                                   Considerations when selecting buffer components.  Additives that increase yield and prevent aggregation and/or
                                   stabilize proteins. Speaker: Dr. Mario Lebendiker (HUJI) (Literature)      17)   15:30 – 16:20    Refolding: Major steps. Protein refolding methods. Screening. Strategies and examples.
                                 Speaker: Dr. Mario Lebendiker (HUJI) (Literature)18)   16:30 – 17:30    The challenge of Membrane protein purification. Main features, use and removal of detergents.
                                  
Speaker: Dr. Mario Lebendiker (HUJI) (Literature)

4th day (18/12/2014)                      

19)   10:00 – 10:50   Advantages and Disadvantages of different expression systems. Connection between expression and
                                    protein quality. Speaker: Prof Gali Prag (TAU) (Literature)

20)   11:00 – 11:50   Purification of protein complexes (case-study: oligomeric chaperone). Speaker: Prof. Abdussalam Azem (TAU)

21)   13:00 – 13:50   Protein-protein interaction. Advantages and disadvantages of SPR, ITC, MST, AUC.
                                  Speaker: Prof. Gideon Schreiber (WIS)

22)   14:00 – 14:50   Protein-protein interaction. Advantages and disadvantages of SPR, ITC, MST, AUC.
                                  Speaker: Prof. Gideon Schreiber (WIS)


23)   15:30 – 16:20   Challenge in the production of biosimilars. Speaker: Dr. Joel Van Gelder (Insight Biopharmaceuticals)

24)   16:30 – 17:30   Major requirements for purification of proteins for structural studies: crystallography, NMR, others.
                                  Speaker: Dr. Mario Lebendiker (HUJI) (Literature)

                                                                                 

                                  Membrane proteins: Practical aspects of over-expressing bacterial membrane proteins for structural studies.

                                  Detergents in crystallography. Speaker: Dr. Mario Lebendiker (HUJI) 

 
Literature Purification Strategy


GE-HEALTHCARE Handbook Protein Purification (pdf)  

GE-HEALTHCARE  Strategies for Protein Purification Handbook  (pdf)  
GE-HEALTHCARE  Protein purification Applications  (pdf)       
GE-HEALTHCARE  Protein and Peptide Purification - Technique selection guide  (pdf)
GE-HEALTHCARE  Purifying Challenging Proteins: Membrane proteins, Multiprotein complexes and Inclusion bodies  (pdf) 
GE-HEALTHCARE  ÄKTAdesign Purification Method handbook. ÄKTAexplorer™ or ÄKTApurifier™ chromatography
(pdf)
GE-HEALTHCARE  Purification of tagged proteins   (pdf)

TOSOH: General Principles of Chromatography (pdf)
Mario Lebendiker Website    Table of  Content    Recommended Links    Selected Protocols 
Latest approaches for efficient protein production in drug discovery. Review - Toshihiko Sugiki et.al. -
        Expert Opin. Drug Discov. (2014) 9(10)   (pdf)
Recombinant protein expression in Escherichia coli: advances and challenges - G.Rosano et. al.-
        Frontiers in microbiology  2014 | Volume5 | Article 172  (pdf)
Challenges and opportunities in the purification of recombinant tagged proteins - A.Pina et. al. -
        Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.12.001  (pdf)
High-throughput production of human proteins for crystallization: The SGC experience - P. Savitsky et al. -
        J. Struct. Biol. (2010), doi:10.1016/j.jsb.2010.06.008  (pdf)
Preparative Purification of Recombinant Proteins: Current Status and Future Trends. Review Article - M. Saraswat -

        (2013) http://dx.doi.org/10.1155/2013/312709  (pdf)

Literature FPLC

GE-HEALTHCARE  ÄKTAdesign Purification Method handbook. ÄKTAexplorer™ or ÄKTApurifier™ chromatography (pdf)

GE-HEALTHCARE  Packing of Gel Filtration column  (movie)

GE-HEALTHCARE  Packing of IEX, Affinity or HIC column  (movie)
GE-HEALTHCARE  Column evaluation (movie)   


Literature IEX
 
GE-Healthcare: Ion Exchange Chromatography & Chromatofocusing  (pdf)
TOSOH: Ion Exchange Chromatography (pdf)
Separation of Protein Charge Variants with Induced pH Gradients Using Anion Exchange Chromatographic Columns
        T. Pabst - Biotechnol. Prog. 2008, 24, 1096 (pdf)

Systematic generation of buffer systems for pH gradient ion exchange chromatography and their application
        F. Kroner - Journal of Chromatography A, 1285 (2013) (pdf)


Literature GF (SEC)

GE-Healthcare: Gel Filtration - Principles and Methods (pdf)

GE-HEALTHCARE  Packing of Gel Filtration column  (movie)
TOSOH: GelFiltration (pdf)

Literature Affinity

GE-Healthcare: Affinity Chromatography: Columns and media  (pdf)  Principles and Methods  (pdf)

GE-Healthcare: Antibody Purification Handbook  (pdf)
Pierce:  Protein Immobilization   (pdf)  
TOSOH
:  Affinity Chromatography resins  (pdf)
EY Lectins  (pdf)
SIGMA-Aldrich Lectins


Literature TAGs & Recomb Proteins

GE-HEALTHCARE: Recombinant Protein Purification Handbook Principles and Methods  (pdf)
GE-HEALTHCARE: GST-Fusion Proteins Handbook  (pdf)

GE-HEALTHCARE:  Purification of tagged proteins   (pdf)                                                                                                                           

Structural Genomics Consortium  Protein production and purification. Nature Methods 2008, 5: 135-146  (pdf-I)  (pdf-II)Purification of Recombinant Proteins - Mario Website
Enhancement of soluble protein expression through the use of fusion tags  - D.Esposito and D. Chatterjee -        Current Opinion in Biotechnology 2006, 17:353–358  (pdf)
Making the most of affinity tags - D. Waugh - TRENDS in Biotechnology Vol.23 No.6 June 2005  (pdf)
Current strategies for the use of aYnity tags and tag removal for the purifcation of recombinant proteins - J.Arnau et al. -         Protein Expression and Purification 48 (2006) 1–13  (pdf)
Advances in recombinant protein expression for use in pharmaceutical research - Assenberg R, et al. -
        Curr Opin Struct Biol (2013), http://dx.doi.org/10.1016/j.sbi.2013.03.008  (pdf)
Review. Production of disulfide-bonded proteins in Escherichia coli - M.Berkmen -  Protein Expression and Purification 82 (2012) 240 (pdf)
Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli
        - J. Klint  et al. - PLoS ONE 8(5): e63865. doi:10.1371/journal.pone.0063865  (pdf)
Statistical approaches to maximize recombinant protein expression in Escherichia coli: A general review - C. Papaneophytou  et al. -       

Protein Expression and Purification 94 (2014) 22–32  (pdf)
Cloning technologies for protein expression and purification - James L Hartley - Current Opinion in Biotechnology 2006, 17:359–366  (pdf)

Literature HIC

Theory and Use of HIC in Protein Purification Applications - Justin T. McCue - Methods in Enzymology (2009) 463 (pdf)
GE-Healthcare: Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods  (pdf)
TOSOH: Hydrophobic Interaction Chromatography  (pdf)
TOSOH: Hydrophobic Interaction Chromatography: Effects of Mixed Electrolytes on Protein Separation  (pdf)

Literature Mixed Mode and RPC
GE-Healthcare: Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods  (pdf)
GE-Healthcare: Multimodal Chromatography Handbook  (pdf)
GE-Healthcare: Purification of influenza A/H1N1 using Capto™ Core 700 (pdf)
VYDAC   The Handbook of Analysis and Purification of Peptides and Proteins by Reverse-Phase HPLC   (pdf)     
Multimodal chromatography: Characterization of protein binding and selectivity enhancement through mobile phase modulators
        L. Wolfe et.al - Journal of Chromatography A, 1340 (2014) 151–156  (pdf)
Multimodal chromatography: An efficient tool in downstream processing of proteins - K. Kallberg et. al - Biotechnol. J. 2012, 7  (pdf)
Evaluation of selectivity in multimodal anion exchange systems: A priori prediction...Y. Hou, S.M. Cramer -
        J. Chromatogr. A (2011)  (pdf)
IgM Purification with Hydroxyapatite - P. Gagnon et. al -  BioProcess International 12(2) February 2014  (pdf)

 

Literature Aggregation
Review: Production of prone-to-aggregate proteins - M.Lebendiker & T.Danieli - FEBS Letters 588 (2014) 236–246 (pdf)

A screening methodology for purifying proteins with aggregation problems.  Lebendiker M., Maes M. and Friedler A. (2015) 

Methods in Molecular Biology: "Insoluble Proteins" book (Springer)1258: 261-281 (pdf)

Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins - D.Leibly et al. -
    PLoS ONE 7(12): e52482. doi:10.1371/journal.pone.0052482  (pdf)
Molecular origins of surfactant-mediated stabilization of protein drugs - H.Lee et al. -  
        Advanced Drug Delivery Reviews (2011) doi:10.1016/j.addr.2011.06.015 (pdf)

Effect of Additives on Protein Aggregation - H.Hamada et al. - Current Pharmaceutical Biotechnology, 2009, 10, 400-407  (pdf)
Protein Aggregation: Pathways, Induction Factors and Analysis - H.Mahler et al. -
        JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 98 (2009)  (pdf)
Mechanisms of Protein Aggregation - J.Philo & and T.Arakawa - Current Pharmaceutical Biotechnology, 2009, 10, 348-351  (pdf)
Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars - S. Berkowitz -  
        NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 527  (pdf)
Interactions of formulation excipients with proteins in solution and in the dried state - S.Ohtake et al. -
        Adv. Drug Deliv. Rev. (2011), doi:10.1016/j.addr.2011.06.011  (pdf)
Classification of Protein Aggregates - L.Narhi et al. - Journal of Pharmaceutical Sciences, Vol. 101, 493–498 (2012)  (pdf)
Protein Misfolding and Aggregation Research: Some Thoughts on Improving Quality and Utility - R. Murphy & C. Roberts
         Biotechnol. Prog., 2013, Vol. 29, No. 5   (pdf)
Strategies for the Assessment of Protein Aggregates in Pharmaceutical Biotech Product Development - J.Engelsman et al. -
        Pharm Res (2011) 28:920–933  (pdf)
Protein Aggregation and Bioprocessing - M.Cromwell et al. -  The AAPS Journal 2006; 8 (3) Article 66 (http://www.aapsj.org). (pdf)
Molecular level insight into intra-solvent interaction effects on protein stability and aggregation - D. Shukla, et al. -

         Adv. Drug Deliv. Rev. (2011), doi:10.1016/j.addr.2011.06.014  (pdf)

Literature Refolding
Protein refolding for industrial processes - E.De Bernardez Clark - Current Opinion in Biotechnology 2001, 12:202–207 (pdf)
Preparative protein refolding - A.Middelberg - TRENDS in Biotechnology Vol.20 No.10 October 2002  (pdf)
Practical considerations in refolding proteins from inclusion bodies - K.sumoto et al. - Prot. Expr. & Purific. 28 (2003) 1–8  (pdf)
In vitro protein refolding by chromatographic procedures - Ming et al. - Protein Expression and Purification 33 (2004) 1–10 (pdf)
On-column protein refolding for crystallization - N.Oganesyan et al. - Journal of Struct. & Funct. Genomics 6: 177–182, 2005 (pdf)

Confronting high-throughput protein refolding using high pressure and solution screens - M.Qoronfleh et al. -
        Protein Expression and Purification 55 (2007) 209–224  (pdf)
Single pH buffer refolding screen for protein from inclusion bodies - B.Coutard et al. -  Protein Expr. & Purific. 82 (2012) 352 (pdf)
Protein refolding using chemical refolding additives - S. Yamaguchi et al. - Biotechnol. J. 2013, 8, 17–31  (pdf)

Literature Membrane Proteins

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and
        Transceptors for Biochemical Studies and Crystallography. - Scharff-Poulsen P, Pedersen PA (2013) -
        PLoS ONE 8(10): e76851. doi:10.1371/journal.pone.0076851  (pdf)
A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes
        - F.Pullara et al.- Protein Expression and Purification 87 (2013) 111–119  (pdf) (pdf Supl)
High-throughput expression and purification of membrane proteins - F.Mancia et al.-
        J. Struct. Biol. (2010), doi:10.1016/j.jsb.2010.03.021  (pdf)
A Fluorescence-Detection Size-Exclusion Chromatography-Based Thermostability Assay for Membrane Protein Precrystallization
        Screening - M.Hattori et al.- Structure 20, 1293–1299, August 8, 2012  (pdf)
An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability -
        J.Fan et al.- Protein Expr. Purif. (2011), doi:10.1016/j.pep.2011.02.010   (pdf)
Detergent selection for enhanced extraction of membrane proteins - B. Arachea et al.-
        Protein Expression and Purification 86 (2012) 12–20  (pdf)
Expression and Purification of Membrane Proteins -J.Kubicek et al.- Methods in Enzymology, Volume 541 (2014)  (pdf)


Literature Protein Quality Control 

Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light
        Scattering - J. Nettleship et al. - (2008) Meth.in Molec.Biol., Vol. 426: Struct.Proteom.: High-throughput Methods  (pdf)
Impact of Residual Impurities and Contaminants on Protein Stability - W.Wang et al. -
        (2014) J.ofPharm.Sc. DOI 10.1002/jps.23931
(pdf)
Building better drugs: developing and regulating engineered therapeutic proteins - Kimchi-Sarfatyet. al.-
        (2013) Trends in Pharmacological Sciences, October 2013, Vol. 34, No. 10  (pdf)
Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars - S. Berkowitz -  
        NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 527  (pdf)

Quality assessment and optimization of purified protein samples: why and how?

Raynal et al. Microbial Cell Factories (2014) 13:180 DOI 10.1186/s12934-014-0180-6 (pdf)Course92632_2014/Quality/Raynal2015.pdf

The Trip Adviser guide to the protein science world: a proposal to improve the awareness concerning the quality of recombinant proteins

Lebendiker M. et al. BMC Research Notes (2014) 7:585  doi:10.1186/1756-0500-7-58  (pdf) (pdf supplement)

 

 

 

HOME
EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS



Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,
The Hebrew University of Jerusalem

mario.l@mail.huji.ac.il  Tel: 972-2-6586920  

Copyright ©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.