1. komponenta

Lecture type	Total
Practicum	60

* Load is given in academic hour (1 academic hour = 45 minutes)

1. Transformation of bacterial cells by recombinant plasmid. Optimization of protein overexpression by variation of the temperature, inductor concentration and time-frame of induction. Checking of the overexpression by SDS-PAGE.
2. Isolation of protein extracts. Purification of the recombinant protein (bearing affinity tag) using affinity chromatography. Fractions analysis by SDS-PAGE.
3. Buffer exchange by dialysis or size exclusion chromatography. Determination of protein concentration. Protein activity test.
4. Isolation of specific protease (like TEV protease) and removal of the affinity tag (like His-tag). Comparison of the activity of the tagged protein with the protein sample where tag is removed.
5. Gel mobility shift assay (analysis of protein-nucleic acid interactions).
6. Pull down (analysis of protein-protein interactions). Silver staining of proteins and nucleic acid. Western-blot analysis.
7. Determination of Kd by fluorescence techniques. This part is optional as it depends on the equipment and fluorophore accessibility. In some case, one group of students can perform these experiments instead of gel mobility shift assay (5) and pull down (6).

LEARNING OUTCOMES:
1. Carry out the experiments related to protein overexpression, protein purification and analysis of noncovalent interactions of proteins with nucleic acids or other proteins.
2. Transform and disrupt bacterial cells.
3. Demonstrate the understanding of various approaches to optimize protein overexpression.
4. Purify overexpressed proteins bearing affinity tag by affinity chromatography.
5. Concentrate proteins, exchange buffer, determine protein concentration, and remove the affinity tags from proteins.
6. Analyze noncovalent protein interactions by electrophoresis under native conditions and by pull down assay

1. Interna skripta za Viši praktikum biokemije (2. radno izd.), Zavod za biokemiju, PMF, Zagreb 2013.
   pET System Manual (11. izd.), Novagen, 2005.
   Relevantna poglavlja iz knjige R. R. Burgess, M. P. Deutscher (ur.), Methods in Enzymology: Guide to Protein Purification (2. izd.), Elsevier, 2009.
   Relevantna poglavlja iz knjige J. R. Lakowicz,Principles of Fluorescence Spectroscopy (3 izd) Springer, 2006.
   Odabrani izvorni i revijalni znanstveni radovi
2. A. Ambriović Ristov (gl. ur.), Metode u molekularnoj biologiji, Institut Ruđer Bošković, Zagreb, 2007.
   J. Sambrook i D. W. Russell, Molecular Cloning: a laboratory manual (3. izd.) Cold Spring Harbor Laboratory Press, 2001.
   S. B. Primrose i R. M. Twyman, Principles of Gene Manipulation and Genomics (7. izd.), Blackwell Publishing, 2006.
   L. Stryer, J. Berg i J. Tymoczko, Biokemija (6. izd), Školska knjiga, 2013. (prijevod na hrvatski jezik)
   J. M. Berg, J. L. Tymoczko i L. Stryer, Biochemistry (7. izd.), W. H. Freeman & Co, New York, 2012.
   D. L. Nelson i M. M. Cox, Lehninger Principles Of Biochemistry (6. izd.), W. H. Freeman & Co, New York, 2013.

Izborni predmeti - Regular study - Molecular Biology