1. Week 6 Review

2. DNA UV Spectra

3. DNA and RNA Bases

4. UV Spectra for Increasing DNA Concentrations

5. UV Spectra for Individual DNA nucleosides

7. Spectra Composition

8. UV Spectroscopy of Nucleic Acids and Proteins 260/280 – DNA ~1.8 – RNA ~2.0 – Lower by 0.2-0.3 acidic solution, protein, phenol, other contaminates – Higher by 0.2-0.3 basic solution 260/280 nucleotides – Guanine 1.15, Adenine 4.5, Cytosine 1.51, Uracil 4.0, Thymidine 1.47

9. UV Spectroscopy of Nucleic Acids and Proteins 230/280 – Higher than 260/280 often in range of 2.0-2.2 – Lower indicates presence of contaminates like phenol that absorb at 280 nm.

10. Example Guanidinium HCl

11. Example- Phenol

12. UV Spectra for ds and ss DNA

13. Why?

14. E. coli Protein Expression Systems Chapter 9 Production of Proteins from Cloned Genes. In Gene Cloning and DNA Analysis and Introduction 2010 by T. Brown.

15. IPTG/DE3 SYSTEM

16. IPTG

17. pET SUMO SUMO Tag Inserted Here

18. SUMO System SUMO Tag is small (~100 residues), heat stable protein with a highly compact globular structure. SUMO Protease 1 is able to selectively cleave fusion proteins, releasing peptides or proteins with any desired N-terminal residue, except proline. SUMO Protease 1 requires the entire SUMO sequence for recognition (recognizes 3D structure) and never cleaves within the fused protein of interest.

19. SUMO Tag Enhances Expression SUMO Tag Chaperones Correctly Folded Proteins SUMO Protease Reliable Cleaver of SUMO Tag SUMO Gene Fusion System

20. IMAC and 6x his Tag Structure pictures taken from Wikipedia

21. IMAC / SUMO System Expression Vector

22. IMAC / SUMO Proteins SUMO Solubility TAG His 6X- Target Gene

23. Steps In IMAC SUMO Production and Purification 1.Clone Gene into SUMO Vector. 2.Express SUMO/Target Gene Fusion Protein in E. coli or other host. 3.Purify SUMO Target Fusion Protein on Ni or Co column (IMAC). Optional (not done in our case) 1.Digest SUMO Target Fusion Protein with SUMO protease. 2.Re-purify Target Protein from SUMO protease and SUMO domain w/6x his tag using Ni or Co column (IMAC).

24. DNA can be a contaminant of a Protein Preparation What would you expect to happen to the UV spectra?

26. PROTEINS

27. UV Spectra of Proteins

28. Protein UV Spectra chem247.files.wordpress.com/2007/09/sept-13-part-iii.pdf‎

30. SDS PAGE Protein is first denatured – Heat unfolds tertiary and secondary structure – Beta-Mercaptoethanol or Dithiotheritol disrupt disulfide cross linkages SDS and Molecular Radius – SDS-coated proteins are linear molecules, 18 Angstroms wide w/length proportional to their molecular weight. – Molecular radius and mobility in the gel is determined by side chains in AA sequence, and the length (i.e. molecular weight of the protein). – SDS-coated proteins have the same approximate charge to mass ratio so no differential migration based on charge.

31. SDS Ensures Protein Remains Denatured During Electrophoresis and Provides Negative Charge