1. PROTEIN TECHNOLOGY By DR ZARINA ZAKARIA

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. Techniques in Protein Technology 1.Isolating 2.Purification 3.Chromatography 4.Electrophoresis

4. Steps in Protein Purification 1.Protein extraction -To prepare protein solution -Total protein only less than 0.1% of total dry weight. -Protein are dissolve in buffer solution -The desired protein is one of many proteins. 1.fractionation 2.chromatography

5. Steps in Protein Purification 2. Fractionation -To separate the crude protein. -Using salting out techniques to precipitate less soluble protein. -Remove one-half to two-third of unwanted protein. -Also using dialysis technique to separate high-molecular-weight and LMW.

6. Steps in Protein Purification 3. 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: 1.gel-filtration chromatography 2.Ion-exchange chromatography 3.Affinity chromatography

7. i-gel filtration chromatography Packed a column with gelatinous polymer that separates molecules according to their molecular size. Molecules larger than gel pores move through the column quickly. Molecules smaller than gel pores move thorough the column slowly. Differences in movement rate make available for separate collection.

8. ii-ion-exchange chromatography Separate proteins on the basis of their charge. Packed column with anion-exchange resins which consist of positively charged materials. Bind protein’s negatively charge groups. Remove protein that do not bind to resin first before recovered the bind proteins.

9. iii-affinity chromatography Uses the unique biologial properties of proteins. A special noncovalent binding affinity between protein and a special molecule (ligand). Ligand is covalent bound to insoluble matrix, which is placed in a column. Nonbinding protein molecule will pass through the column. Binding protein removed by altering the conditions that affect binding.

10. 4-Electrophoresis Protein are electrically charge, so they move in electrically field. 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.

11. Electrophoresis-continue Electrophoresis carry out by using gels such as polyacrylamide or agarose. Similar in function to gel-filtration chromatography but more effective.

12. 6-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. 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.

13. 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.

14. Protein characterization-continue 4-determination of the sequences of the peptide fragments. 5-ordering the peptide fragments Molecular weight determination Methods used are: i-gel-filtration column chromatography ii-SDS PAGE iii-ultracentrifugation

15. Molecular weight determination There is correlation between molecular weight and elution volume, Ve. Elution volume is the volume of solvent required to elute the protein from the column since it first contacted the gel. The MW of the protein is determined by comparing its relative elution volume (Ve- Vo/Vg) to those several standard molecules.

16. Molecular weight determination- continue Sodium dodecyl sulfate (SDS) is a powerful negatively charge detergent. Polyacrylamide gel electrophoresis (PAGE) is to separate protein base on MW. Ultracentrifugation uses high centrifugal forces to determine the protein MW.

17. X-Ray Crystallography To obtain information on three- dimensional structure about protein. Use electromagnetic radiation to resolve protein structure. Crystalline specimen are exposed to X-ray beam to produce a scattered atoms in the crystal. The scattered image is reconstructed mathematically to get the structural model.

18. References 1.McKee, T. & McKee, J.R. Biochemistry: The molecular Basis of Life.McGraw Hill 2.Horton, Moran, Scrimgeour, perry & Rawn. Principles of Biochemistry. Pearson.