FE 520

PROTEIN PURIFICATION

Instructor:  Prof. Dr.  Şebnem Harsa

                     Office: (232) 750 62 91

                     E-mail: sebnemharsa@iyte.edu.tr

Prerequisite(s):

 Basic knowledge of microbiology and biochemistry is necessary.

 Text Books:

 There is no required textbook for the course, lecture notes and supplemental material are available.

 Reference Books:

 Janson, J.C. & Ryden, L. Protein Purification: Principles, High Resolution Methods and Application. New York: VCH.

Wheelwright, S. M. Protein Purification: Design and Scale Up of Downstream Processing. John Wiley & Sons.

Deutscher, M.P. Guide To Protein Purification. San Diego, New York: Academic Press.

Harris, E.L.V. & Angal, S. Protein Purification Applications: A Practical Approach. Oxford: IRL Press.

Scopes, R.K. Protein Purification: Principles and Practice. Springer Verlag.

 Course Description:

 This course provides students with a broad overview to the high resolution, affinity and selective purification techniques that are commonly used in protein purification. Lectures cover the theoretical and practical aspects of protein purification methods in addition to discussing limitations of various procedures.  The course consists of cell disintegration, precipitation and salting out, gel filtration, ion-exchange, reversed phase, hydrophobic interaction and affinity chromatography methods. Understanding aqueous two phase systems, reversed micelles, liquid membranes, electrodialysis, electrophoretic methods and isoelectric focusing of proteins are also in the scope of lectures. At the end of the course the students are expected to have a better understanding about laboratory and large scale protein purification techniques.

 Course Outline:

 I.                    Introduction

Impact of upstream operations and strategies for downstream process design

An overview of fractionation techniques

Fractionation strategies

Monitoring the fractionation

The final product

Laboratory equipment                              

II.                 Making an Extract

The raw material

Cell disintegration and extraction

Optimization and clarification of the extract

Procedures for particulate associated enzymes

III.               Separation of Particulate Material

Filtration

Centrifugation

IV.              Separation by Precipitation

The solubility of protein at low salt concentration, isoelectric precipitation

Salting out at high salt concentration

Precipitation with organic solvents

Precipitation with organic polymers and other materials: affinity precipitation

Precipitation by selective denaturation

V.                 Separation by Adsorption

General chromatography theory

Batch adsorption

High performance liquid chromatography (HPLC)

VI.              Gel Filtration Principles

Media for separation

Applications 

VII.            Ion-Exchange Chromatography

Theory

Applications

Factors influencing separation behaviour

VIII.         Hydrophobic Interaction Chromatography

Theory

Applications

Factors influencing separation behaviour 

IX.              Affinity Chromatography

Theory

Applications

Factors influencing separation behaviour 

X.                  Electrophoretic Methods

Principles

Applications

XI.               Liquid Membrane Separations

Emulsion Liquid Membranes

Supported Liquid Membranes

Reversed Micellar Extraction

Theory and Applications

XII.             Large-Scale Protein Purification Examples; Design, Scale-Up and Economics

Teaching Methods

 The topics are given by the instructor using power point presentations. A seminar is given by the students as the term project.

Lectures: 3 hrs/week for 12 weeks

3-hours written midterm examination: 25% during regular midterm examination period of the

     university calendar

3-hours written final examination: 50% during regular final examination period of the university

    calendar

Seminar presentation: 25%

 Grading

Midterm Exam: 25 %        

Final Examination: 50%      

Presentation: 25%

 Study Material

 Dziembowski, A. & Seraphin, B. “Recent developments in the analysis of protein complexes”, FEBS Letters 556: 1-6, (2004).

 Jungbauer, A. “Chromatographic media for bioseparation”, J. of Chromatography A. 1065: 3- 12, (2005). 

Guiochon, G. “Preparative Liquid Chromatography”, J. of Chromatography A. 965: 129-161 (2002). 

Winsor, D.J. “Analytical exclusion chromatography”, J. of Biochemical and Biophysical Methods, 56: 15-52 (2003).

 Konstanski, L.K., Keller, D.M. & Hamielec, A.E. “Size-exclusion chromatography: a review of calibration methodologies”, J. of Biochemical and Biophysical Methods, 58: 159-186 (2004).

 Irvine, G.B. “Size-exclusion high performance liquid chromatography of peptides: a review”, Analytica Chimica Acta, 352: 387-397, (1997).