1. Goals for Monday 2/18 1.Induce, harvest, and lyse BL21DE3 cells expressing acetate kinase or acetate kinase variant (manual pg..) 2.Lifetime of protein lab 3.Lecture 4.Measure protein and cytochrome in previous purification

2. We can “control” protein expression With the notable exception of proteins such as those that compose the ribosome, many proteins are found only in low abundance (particularly proteins involved in regulatory processes) Thus, we need to find ways to grow cells that allow ample expression of proteins that would be interesting for biochemical characterization.

3. Find conditions for cell growth that enhance a protein’s expression For example, cytochrome c 2 is utilized by R.sphaeroides for both respiratory and photosynthetic growth; a slight increase in levels of this protein is observed under photosynthetic growth conditions. However, Light-Harvesting complexes are only synthesized under photosynthetic growth conditions; obviously if you want to purify this protein you need to grow cells under photosynthetic conditions

4. Molecular Biology allows us to manipulate genes  Understanding the basic mechanisms of gene expression has allowed investigators to exploit various systems for protein expression  Prokaryotic expression systems  Eukaryotic expression systems Yeast Mammalian  Viral expression systems Baculovirus and Insects

5. Proteins are encoded by genes M—P—Q—Q--STOP PROTEIN (PEPTIDE) 5’-GATGCCCCTCGAATAA-3’ 3’-CTACGGGGAGCTTATT-5’ DNA 5’-GAUGCCCCAGCAAUAA-3’ mRNA Predicted genes or genes of unknown function are typically called open reading frames (ORF’s)

7. What do we need to produce a protein? lamB A gene lamB Promoter Transcriptional unit Terminator lamB Ribosome binding site Translational unit

8. Molecular Biology presents an opportunity for useful genetic constructs lamB Promoter Terminator bla Plasmid ori Antibiotic resistance gene Origin of Replication Can fuse gene to other sequences conferring affinity

9. Choice of promoter allows control over transcription levels  Intrinsic promoters can be sufficient for overexpression in multi-copy plasmids  Constitutive promoters with high activity (ie. promoters for ribosomal genes) can be useful for producing non-toxic proteins  Inducible promoters allow control of expression, one can “titrate” the promoter activity using exogenous agents

10. An expression system utilizing lactose and T7 RNA polymerase is a popular choice in prokaryotes lamB bla ori Plasmid T7 polymerase dependent promoter T7 pol Lactose-inducible promoter Genome

11. Inclusion bodies provide a rapid purification step Proteins exist as aggregates in inclusion bodies thus special precautions must be taken during purification. Typically, inclusion bodies can be readily isolated via cell fractionation. following isolation the proteins must be denatured and renatured to retrieve active protein.

12. Additional concerns regarding protein expression Modifications Inclusion bodies Codon usage

13. Cells exhibit nonrandom usage of codons This provides a mechanism for regulation; however, genes cloned for purposes of heterologous protein expression may contain “rare” codons that are not normally utilized by cells such as E. coli. Thus, this could limit protein production. Codon usage has been used for determination of highly expressed proteins.

14. Molecular Biology allows us to manipulate genes  Understanding the basic mechanisms of gene expression has allowed investigators to exploit various systems for protein expression  Prokaryotic expression systems  Eukaryotic expression systems Yeast Mammalian  Viral expression systems Baculovirus and Insects

15. Non-prokaryotic expression systems have emerged due to increasing simplicity and the need for proper modifications. Although you can express a eukaryotic cDNA in a prokaryote is the protein you purify, what the eukaryotic cell uses? Invitrogen : www.invitrogen.comwww.invitrogen.com Gateway vectors Novagen: www.novagen.comwww.novagen.com

16. http://www.the-scientist.com/yr1997/sept/profile2_970901.html Considering expression systems? http://www.biochem.wisc.edu/biochem660/pdfs/readings/lecture02/Larsen2.pdf http://www.baculovirus.com/ http://www.biowire.com/bw_jsp/home_top.jsp http://biobenchelper.hypermart.net/pr/expression.htm

17. Goals for Tuesday 2/19 1.Lecture 2.Load and run SDS-PAGE gel for cytochrome purification 3.Prepare crude extracts (centrifugation) 4.Purify His-tagged proteins 5.Generate standard curve for acetyl-phosphate determinations (pg…)

18. Several hyperthermophilic archaeal species have also been shown to be dependent on tungsten (W), also Cd important in diatoms

19. Fe is most abundant, followed by Zn

20. Metals in Biology All ribozymes are metalloenzymes, divalent cations are required for chemistry, and often aid in structural stabilization. Protein enzymes are divided into six classes by the Enzyme Commision: 1.Oxidoreductase 2.Transferase 3.Hydrolase 4.Lyase 5.Isomerase 6.Ligase Zn is the only element found in all of these classes of enzymes.

21. Proteins bind metals based on size, charge, and chemical nature Each metal has unique properties regarding ionic charge ionic radii, and ionization potential Typically, metals are classified as “hard” or “soft” in correlation with their ionic radii, electrostatics, and polarization Hard metals prefer hard ligands, soft prefer soft, Borderline metals can go either way.

22. Properties of metal ions determine their biological utility

23. Soft Hard

24. Metals favor distinct coordination in proteins M L L L L M L L L L M L LL L L L M L L L L L Tetrahedral Square Planar Trigonal bipyramidal Octahedral M = Metal L = Ligand

25. Unsaturated coordination spheres usually have water as additional ligands to meet the favored 4 or 6 coordination

26. Protein sequence analyses have revealed certain metal binding motifs Structural Zn are generally bound by 4 cysteines Catalytic Zn bound by three residues (H, D, E, or C) and one water Coordination in primary sequence of alcohol dehydrogenase Catalytic L 1 -few aa-L 2 -several aa-L 3 Structural L 1 -3-L 2 -3-L 3 -8-L 4 L = Ligand

28. Biological roles of transition metals  Coordination Structure (protein and protein-substrate)  Electrophilic catalysis Positive charge attracts electrons, polarize potential reactant, increase reactivity  General Acid – Base catalysis  Redox reactions  Metalloorganic chemistry Free radicals (not just limited to proteins*)

29. Carbonic Anhydrase catalytic mechanism

30. Molybdenum?? http://www.dl.ac.uk/SRS/PX/bsl/scycle.html

31. Tetrapyrroles (heme, chlorophyll) make proteins “visible” along with certain metals

32. Spectroscopy is a study of the interaction of electromagnetic radiation with matter A =  cl Absorbance = extinction coefficient x concentration x path length Beer-Lambert Law The amount of light absorbed is proportional to the number of molecules of the chromophore, through which the light passes Units: None = M -1 cm -1 M cm

33. c-type cytochromes have a characteristic absorbance spectrum Isobestic point

34. Purification of cytochrome c 2 overview  Cell Fractionation  Protein precipitation  Hydrophobic Interaction chromatography  Gel electrophoresis  Optical spectroscopy

35. Lab reports Introduction – Rationale for why these experiments are important (not simply from a course work perspective) Materials & Methods – Concise, but detailed description of how experiments were performed Results – Summary of data (Simply report data, ie. purifica- tion table, etc.) Discussion – Implications of results All lab reports must be type-written (please)

36. Keeping a purification table

37. SDS-PAGE examination of purification 1 2 3 4 1 – Molecular weight markers (see below) 2 – Periplasm fraction 3 – Ammonium sulfate fraction 4 – Phenyl Sepharose fraction MW marker sizes: 97.4 kDa 66.2 kDa 45 kDa 31 kDa 21.5 kDa 14.4kDa Explain these results in your lab report

38. Periplasmic Fraction Ammonium Sulfate Fraction Phenyl Sepharose Fraction Used 1.0 ml of Ammonium Sulfate Fraction and phenyl sepharose fraction; Used a 1:10 dilution of periplasmic Fraction for these readings