Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8“ ‘omics “Protein PurificationColumn ChromatographySlide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Importance of Overexpression in Protein PurificationClicker QuestionSlide 21Slide 22DNA SupercoilingSlide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Relaxing DNATwo Types of TopoisomerasesSlide 33DNA Gyrase is a special Type II TopoisomeraseSlide 35Two Types of TopoisomerasesType I TopoisomeraseSlide 38Slide 39Supercoiling Affects Electrophoretic MobilitySlide 41Clicker QuestionSlide 43All Cells Require TopoisomerasesPATHWAYS TO HEALTH CAREERS Thu. Sept. 18, 2014 4PM-5:30PM 161 NOYESThe MCB Advising Program will host health care professionals and MCB Alumni for a panel discussion on Pathways to Health Careers. Students will hear from doctors regarding their personal journey into a health career. Attendees are encouraged to come with any and all questions relating to health careers. Our alumni are very open and candid in their responses.Richard Berkowitz, MD, Chairman and Medical Director, Dept. of Anesthesiology, Community Hospital, Munster, IN. Clinical Assoc. Professor of Anesthesiology and Pediatrics, UIC and Volunteer Clinical Asst. Professor, Indiana University School of Medicine.Kawhar Siddique, MD, CEO of Beverly Hills Spine Surgery and an Attending Neurosurgeon at Cedars-Sinai Medical Center in Los Angeles. Rachel M. Frank, MD is a 4th year resident in Orthopedic Surgery at Rush University Medical Center. Barry J. Riskin, MD , Neurologist affiliated with Presence Covenant Medical Center and Carle Foundation Hospital.Sept. 18, 2014Resume Blitz 140 Burrill HallUndergraduate students 6:30 pm – 8 pm (Panel 6:30 pm – 7:00 pm; Resume Critique 7:15 pm – 8:00 pm) Sept. 25, 2014Career Fair Prep/Networking SessionHeritage Room ACES Library5:30 pm – 7 pm Oct. 2, 2014Recruiters’ Interviewing Tips1128 Foreign Language Building5:30 – 7:00 pmSessions led by employers!Pre-Career Fair WorkshopsMCB 250 Exam IWednesday September 17th 7:00-9:00 PMSection Teaching Assistant RoomADQ, ADR, ADS, ADT, ADU, ADX, ADY, ADZSunetra Biswas, Preethi Ragunathan141 Loomis LaboratoryADE, ADF, ADG, ADH William Arnold 151 Loomis LaboratoryADA, ADB, ADC, ADV, ADM, ADOXinyun Cao, Michael Tencati114 David Kinley HallADI, ADJ, ADK, ADL, ADNTulip Mahaseth, Koh-Eun Narm100 Materials Science and Engineering (MSEB)MCB 250 Lecture 9Protein PurificationDNA SupercoilingChromosome Structure2-D PAGE - proteins associated with the cell membrane of the organism that causes Lyme diseaseMol. Wt. (kDa)pH 11pH 2.5Proteomics - the study of large groups of proteins. The “proteome” - all of an organism’s proteins.•All of the spots on the previous 2-D gel can be identified.–Pick spots, then digest with trypsin (a protease that cuts only at Lys and Arg residues). This can be done robotically.–Mass spectrometry can determine the masses of all the peptides produced from each spot very precisely–The genome DNA sequence of the organism is known so the computer can compare the masses of the peptides from each spot with masses of the peptides expected from gene’s coding region.–The gene encoding each spot can then be identified.“ ‘omics “•Genome - the complete DNA sequence of an organism - human has about 20,000 genes but much more DNA.•Transcriptome - the complete set of RNA molecules transcribed from a genome - size of human transcriptome unknown but very large.•Proteome - the complete set of proteins - about 100,000 proteins in the human.•Metabolome - the complete set of small molecules found in a cell.Protein Purification•To study individual proteins in detail you need relatively large amounts (milligrams) of pure proteins.•Goal of purification is to obtain a pure, undenatured protein from a mixture that may contain hundreds or thousands of different proteins.•Various techniques are used to separate proteins based on their properties, usually chromatography based on one of the following:–Size–Charge–Hydrophobicity–Specific Binding•You MUST have some assay to follow your protein through the purificationColumn ChromatographyChargeShape (Size)Specific BindingSeparation Based on Charge: Ion Exchange ChromatographyThe positively charged protein sticks to the beads, the negatively charged protein does not. The postiviely charged protein can be eluted from the column by increasing the concentration of salt in the elution solution.Fig 7-26Separation Based on Size: Size Exclusion or Gel Filtration ChromatographyThe beads contain aqueous pores. Smaller proteins can “explore” the spaces, slowing their progress through the matrix. Larger proteins are excluded from some or all of the spaces – they elute more quickly.Fig 7-26Multistep Protein Purification – Protein Specific-Trial and ErrorSeparation based on Affinity: Affinity ChromatographyIdea: If you could put a tag on a protein that makes it bind tightly to something that proteins do not normally bind to, you could separate the tagged protein from all the other proteins in a mixture of proteins.How could you do this? Use molecular cloning.DNA encoding the protein you want to purifyRegion of vector encoding 6 histidines- a “His-tag” (in red)This plasmid will produce a new protein: the protein you want to purify with 6 His residues at its C-terminal.LigationPlasmid VectorSo what does the His-tag do for you?Ni2+ affinity column. Affinity ChromatographyBacterial cell containing the plasmid encoding the His-tagged proteinYour proteinHis-tagThe His-tag chelates (binds to) Ni2+ ions.Affinity ChromatographyNi ColumnCrudeextractEluent- Competes with the His-tag for Ni2+ resulting in release of the tagged proteinPureProteinBacterial cell producingHis-tagged proteinSDS-PAGE can be used to follow purification of a protein.Importance of Overexpression in Protein Purification•Many proteins in the cell are present at very low concentration. Important proteins may comprise 0.1% or less of the total protein in the cell•By cloning the gene encoding the protein you wish to purify, you can usually greatly increase the level of the protein. Plasmids may be present in many copies, amplifying the gene encoding your protein. It’s much easier to purify a protein that is 10% of the total protein than one that is 0.1%.•Using cloning, proteins can be expressed in and purified from organisms that are convenient for purification. A human protein might