ERT106 BIOCHEMISTRY PROTEIN TECHNOLOGY

1. ERT106 BIOCHEMISTRYPROTEIN TECHNOLOGY By Pn Syazni Zainul kamal

2. Why to exploit protein?? • Information about protein structure has led to a deeper understanding of the evolutionary relationships between species. Exp. To differentiate two species of Mycobacterium name as M. gastri and M. kansasii. • Some inherited disease caused by alterations in the amino acid sequence of specific protein. Exp. To detect mutated protein that caused of inherited Parkinson's disease.

3. Step to isolate typical protein • preparation: determination sources, assay method • Extraction : crude extract (mix with other protein) • Purification : step by step purification (salting out, dialysis, chromatography) • Characterization : determination of amino acid composition

4. 1) Preparation • To develop an assay for the protein of interest • Assay must be specific for the protein of interest • Assay must be convenient to perform coz it will be used frequently during investigation

5. 2) Extraction • Depending on the source • the protein has to be brought into solution by breaking the tissue or cells containing it. • Can be achieve through : homogenization, sonication, repeated freezing and thawing • Fraction of protein are dissolved in buffer solution

6. 3) Purification • Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. • 1st step = salting out/precipitation is a technique in which high concentration of salts eg.ammonium sulfate (NH4)2SO4 are used to precipitate protein • This technique removes many impurities (Remove one-half to two-third of unwanted protein)

7. Dialysis is used to removed the salt • Result = protein become more pure and more sophisticated methods are used to archive further purification ie. Chromatography & electrophoresis

8. Chromatography • To further fractionate mixture of proteins that remains after salting-out and dialysis. • To separate protein mixtures on the basis of molecular properties such as size, shape, and weight or certain affinities. • 3 types of chromatographic methods commonly used are: • gel-filtration chromatography • Ion-exchange chromatography • Affinity chromatography

9. Often several techniques must be used sequentially to obtain a demonstrably pure protein • In all chromatographic method : - protein is dissolved in a liquid (mobile phase) eg. Buffer - as protein mol. pass across the stationary phase (a solid matrix), they separate from each other

10. gel filtration chromatography • A column packed with gelatinous polymer that separates molecules according to their size & shape. • Molecules larger than gel pores move through the column quickly. • Molecules smaller than gel pores diffuse in and out the gel pores, so their movement through the column is retarded.

13. The smaller the molecular weight, the slower the movement • Differences in movement rate make available for separate collection.

14. ion-exchange chromatography • Separate proteins on the basis of their charge. • Packed column with : a) anion-exchanged resin (+ve charged materials), -ve charged protein will bind or b) cation-exchanged resin (-ve charged materials), +ve charged protein will bind • Remove protein that do not bind to resin first before recovered the bind proteins with an appropriate change in the solvent pH/salt concentration

16. Remove protein that do not bind to resin first before recovered the bind proteins with an appropriate change in the solvent pH/salt concentration

17. affinity chromatography • Uses the unique biological properties of proteins. • A special noncovalent binding affinity between protein and a special molecule (ligand). • Ligand is covalenty bound to insoluble matrix, which is placed in a column. • Nonbinding protein molecule will pass through the column first. • Binding protein molecule is removed by altering the conditions that affect binding. (pH, salt conc)

19. Electrophoresis • Protein are electrically charge, so they move in electric field. • Electrophoresis = Molecules separate from each other because of differences in their net charge. • Molecules with +ve net charge migrate toward –vely charge electrode (cathode). • Molecules with no net charge will not move at all.

20. Electrophoresis carry out by using gels such as polyacrylamide or agarose (separate the protein on the basis of their molecular weight) • Similar in function to gel-filtration chromatography but more effective.

22. 4) Protein characterization • By knowing the amino acid composition and amino acid sequencing. • AA composition is accomplish by determining the number of each type of amino acid residue present in the molecule. • Begin with complete hydrolysis of all peptide bonds then analysis of resulting amino acid mixture

23. Compose of many process such as: i) hydrolysis of all peptide bonds with 6N HCL for 10-100 hours. ii) analysis of resulting amino acid mixture or hydrolysate by using ion-exchange chromatography or HPLC.

24. Protein characterization-continue • AA sequencing is to determine a protein’s primary structure. • Involves several steps: 1) cleavage of all disulfide bonds. 2) determination of the N-terminal and C-terminal amino acids. 3) cleavage of the polypeptide into fragments.

25. Protein characterization-continue 4) determination of the sequences of the peptide fragments. 5) ordering the peptide fragments