Protein Production for Biophysical and Biochemical Studies -PPBBS (#92632)
                                                                    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 optimization

Ø  Genetically engineered Tags. Cleavage Sites

Ø  Additional techniques use in protein production

Ø  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
    

1^st day (06/11/2014)

1)     10:00 – 10:50   Introduction to Protein Purification. Main stages. (Literature)

2)      11:00 – 11:50   FPLC system. FPLC software  

3)      13:00 – 13:50   FPLC system. (Literature)

4)      14:00 – 14:50   Ion Exchange: Main stages in chromatography. Basis for selectivity. Operational considerations. Determination of
                                   start conditions. Parameters for optimization. (Literature)

5)      15:30 – 16:20  Ion Exchange(continue), examples

6)      16:30 – 17:30 Hadar Protein concentration: native methods, denaturative methods. Protein determination: different methods.    
                                 Dialysis. Ultrafiltration.

2^nd 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. (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. (Literature)                  

9)      13:00 – 13:50   Affinity chromatography: Main stages. Advantages and Disadvantages. Type of Affinities.
                                   Designing and preparing an affinity gel. (Literature)

10)     14:00 – 14:50   Affinity chromatography: Genetically engineered Tags, recombinant proteins. Cleavage Sites.
                                   Parameters for optimization. Examples. (Literature)

11)     15:30 – 16:20  Other Purification Techniques: Hydroxyapatite, Reverse Phase, Multimode Resins (HIC + IEX & GF + IEX)  (Literature)

12)     16:30 – 17:30 Ofrah Additional techniques use in protein production  Electrophoresis: Native and denaturative gels; Sample buffer
                                (+/- reducing agents or 5’ 100C); stain (sensitivity); western (information we get, etc).                        

3^rd day (04/12/2014)

13)   10:00 – 10:50   Cell disruption considerations. Centrifugation. Filtration.  Columns, membranes.

14)   11:00 – 11:50   Gali Prag Advantages and Disadvantages of different expression systems. (Literature)
                                   Connection between expression and protein quality

15)   13:00 – 13:50   Input for Purification Protocol Development. Guidelines for Protein Purification.

16)   14:00 – 14:50   Selection and combination of purification techniques. Storage

17)   15:30 – 16:20   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. (Literature)                  

18)   15:30 – 16:20 Refolding: Major steps. Protein refolding methods. Screening. Strategies and examples. (Literature)

19)   16:30 – 17:30 The challenge of Membrane protein purification. Main features, use and removal of detergents. (Literature)

4^th day (18/12/2014)                      

20)   10:00 – 10:50  Membrane proteins: Practical aspects of over-expressing bacterial membrane proteins for structural studies.
                                  Detergents in crystallography. (Literature)

21)   11:00 – 11:50   Abdussalam Azem Purification of protein complexes (case-study: oligomeric chaperone)

22)   13:00 – 13:50  Gideon Schreiber Protein quality: Light Scattering, SEC-MALS. Circular dicroism.

23)   14:00 – 14:50  Gideon Schreiber Protein-protein interaction. Advantages and disadvantages of SPR, ITC, MST, AUC

24)   15:30 – 16:20  Major requirements for purification of proteins for structural studies: crystallography, NMR, others.

25)   16:30 – 17:30  Orna Dreazen  Protein quality requirements in the industry: Protein assays, methods and limitations. Electrophoresis.
                                  Analytical chromatography (SEC, IEX, RPC).

Literature HIC

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)
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)