Genetik Mühendisliği - Hacettepe Üniversitesi
advertisement
BYM613 Genetik Mühendisliği Hacettepe Üniversitesi Biyomühendislik Bölümü 2012-2013 Güz Dönemi Dr. Eda Çelik-AKDUR [email protected] Proje Sunumları: 8 Ocak, 2013 Salı 13:15-16:30 Proje Raporu Teslim: 10 Ocak 2013, Perşembe 1 Proje’de değerlendirilmesi beklenen konular : •Ürününüzü, ürünün yapısını (örneğin PTM var mı), E.C. numarasını, ekonomik değerini, kullanım alanlarını açıklayınız. (BRENDA web sitesinden faydalabilirsiniz.) •Rekombinant olarak üreteceğiniz mikroorganizmayı ve özelliklerini, neden bu organizmayı seçtiğinizi tartışınız. •Proteini kodlayan geni hangi organizmadan nasıl elde edeceğinizi veya hangi gen bankasından satın alacağınızı belirtiniz. Gen bankası numarasını belirtiniz (NCBI web sitesinden bulabilirsiniz.) •Üretim için klonlayacağınız vektörü, türünü, nereden satın alacağınızı (örneğin Invitrogen, ATCC, vs.), neden bu vektörü seçtiğinizi açıklayınız. •Proteinin nereden (Periplazmadan, sitoplazmadan veya hücre dışından) saflaştırılacağını ve önerdiğiniz saflaştırma basamaklarını 1 paragrafta açıklayanız (Ör: önce hücreler çöktürülecek, sonra hücre atılacak veya hücre içi ise parçalanacak, daha sonra örneğin affinite kromatografisi ile saflaştırılacak, vs.). Vektörün tasarım aşamalarını detaylandırınız. • Örneğin hangi promotörü içeriyor ve bu güçlü mü, yoksa zayıf bir promotör müdür? • Genin kaynağını biliyorsunuz, peki nasıl çoğaltacaksınız? (PCR ile mi, yoksa hücreleri dolayısıyla plazmidi çoğaltıp, plazmidi ekstrakte edip, RE ile geni kesmek suretiyle mi?) PCR ise primerlerinizi tasarlayınız. • Hangi RE’lerini kullanarak vektör ile birleştireceksiniz. Bu enzimlerin çalışma koşulları nedir? (çoğu enzimin optimum çalışma sıcaklığı 37°C’dir ancak kontrol edilmelidir.) • Proteini saflaştırmak veya ucuz antikorlar ile görüntüleyebilmek için (Western Blot ile), vektörünüze ilgili peptidi kodlayan bir DNA dizini ekleyecek misiniz? Bu dizini, PCR ile ancak ekleyebilirsiniz, dolayısıyla PCR için primerlerinizi tasarlarken bunu da göz önüne almalısınız. • Vektörün içine klonlayacağınız kasetin DNA dizinini, FASTA formatında ve ilgili bölgeleri adlandırılmış biçimde raporunuza ekleyiniz. (kaset: geni, RE bölgelerini, saflaştırma/tanıma peptidlerini kodlayan DNA dizinini içerir. Ayrıca, vektörde bulunmuyorsa, başlama ve dur kodonlarını da içermelidir.) 2 “Proteomics” & “Metabolomics” •Proteomics- is the study of the proteome, which corresponds to all of the proteins expressed in a given tissue under a particular set of conditions •Metabolomics-is the study of metabolites (e.g., calcium, cholesterol, glucose) present in a given tissue under a particular set of conditions * Others now such as Bioinformatics and Glycomics 3 “Genomics” a branch of biotechnology concerned with applying the techniques of genetics and molecular biology to the genetic mapping and DNA sequencing of sets of genes or the complete genomes of selected organisms, with organizing the results in databases, and with applications of the data (as in medicine or biology) Anticipated Benefits of Genome Research Molecular Medicine • improve diagnosis of disease • detect genetic predispositions to disease • create drugs based on molecular information • use gene therapy and control systems as drugs • design “custom drugs” (pharmacogenomics) based on individual genetic profiles Microbial Genomics • rapidly detect and treat pathogens (disease-causing microbes) in clinical practice • develop new energy sources (biofuels) • monitor environments to detect pollutants • protect citizenry from biological and chemical warfare • clean up toxic waste safely and efficiently U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 4 Anticipated Benefits of Genome Research-cont. Risk Assessment • evaluate the health risks faced by individuals who may be exposed to radiation (including low levels in industrial areas) and to cancer-causing chemicals and toxins Bioarchaeology, Anthropology, Evolution, and Human Migration • study evolution through germline mutations in lineages • study migration of different population groups based on maternal inheritance • study mutations on the Y chromosome to trace lineage and migration of males • compare breakpoints in the evolution of mutations with ages of populations and historical events U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 Anticipated Benefits of Genome Research-cont. DNA Identification (Forensics) • identify potential suspects whose DNA may match evidence left at crime scenes • exonerate persons wrongly accused of crimes • identify crime and catastrophe victims • establish paternity and other family relationships • identify endangered and protected species as an aid to wildlife officials (could be used for prosecuting poachers) • detect bacteria and other organisms that may pollute air, water, soil, and food • match organ donors with recipients in transplant programs • determine pedigree for seed or livestock breeds • authenticate consumables such as caviar and wine U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 5 Anticipated Benefits of Genome Research-cont. Agriculture, Livestock Breeding, and Bioprocessing • grow disease-, insect-, and drought-resistant crops • breed healthier, more productive, disease-resistant farm animals • grow more nutritious produce • develop biopesticides • incorporate edible vaccines incorporated into food products • develop new environmental cleanup uses for plants like tobacco U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 Medicine and the New Genetics Gene Testing Pharmacogenomics Gene Therapy Anticipated Benefits: • improved diagnosis of disease • earlier detection of genetic predispositions to disease • rational drug design • gene therapy and control systems for drugs • personalized, custom drugs U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003 6 Protein techniques Protein Identification Protein Expression Protein Purification Protein-Protein interactions Application in literature 7 Methods Used for Protein Quantity Determination Biuret Test Folin-Ciocalteu ( Lowry ) Assay Bicinchoninic Acid ( BCA ) Assay Dye-Binding ( Bradford ) Assay Ultraviolet Absorbance Ultraviolet Absorbance Estimation Procedure 1. Zero spectrophotometer to water (or buffer) 2. Take the absorbance at 280 nm in a quartz cuvette 3. Change wavelength to 260 nm and zero with water (or buffer) 4. Take absorption at 260 nm in a quartz cuvette 5. Use the following equation to estimate the protein concentration [Protein] (mg/mL) = 1.55*A280 – 0.76*A260 8 Protein Identification Sequencing (Edman degradation) Centrifugation (cellular location) 1D/2D Gel Electrophoresis ELISA “Enzyme-linked immunosorbent assay” Mass spectrometry Determine approx the first 20 AA Break sample into peptides Molecular mass is determined using mass-to-charge (m/z) ratios of ions AA sequence can be determined Protein microarray NMR (Nuclear Magnetic Resonance) X-Ray Cristallography What You Can Learn from Amino Acid Sequence 1. Molecular weight of the polypeptide chain 2. Charge versus pH; Isoelectric point 3. Hydrophobicity & membrane spanning regions 4. Potential modification sites 5. Conserved motifs that suggest cofactor affinity What You Can’t Learn from Amino Acid Sequence 1.Function 2. 3-Dimensional structure; Shape 3. Multi-subunit features 4. Ammonium sulfate precipitation properties 5. Surface features (hydrophobic patches, charge distribution, antigenic sites) 9 Differential centrifugation Gel electrophoresis Denaturing – SDS-PAGE Non-denaturing SDS gives uniform neg. charge Separates proteins by size/mass Separates based on charge and size/conformation Often combined with Western blotting (using antibodies specific for proteins of interest) 10 SDS-PAGE 1 2 3 4 5 6 2D gel electrophoresis 1st Separation based on pI isoelectric focusing of zwitterions 2nd dimension dimension Normal SDS-PAGE * pI = pH where protein has zero net charge Typical range of pI = 4-9 11 Estimating the charge of a protein At pH 3 the protein will be +ve [H+] pH ~3 pI ~5 [OH-] Protein becomes increasingly +ve [H+] [OH-] Protein becomes increasingly -ve 2D-GE 12 Western Blotting Mass spectrometry IDs based on mass-to-charge (m/z) ratio Samples are broken down and analyzed Proteins -> peptides Able to determine seq of peptides Database search to ID protein 13 Mass spectrometry 14 Mass spec combos LC/MS MALDI-TOF MS Liquid chromotography to separate peptides Matrix-assisted laser desorption-time of flight Samples are ionized and “flight time” through an electrified tube is measured Tandem MS Multiple MS measurements on a single sample Identifies peptide sequence ELISA Enzyme-linked immunosorbent assay 15 Protein Microarray Kullanım alanları •proteinlerin varlığının ve miktarının belirlenmesi •protein-protein etkileşimi •protein-nükleik asit etkileşimi •enzimlerin substratlarının bulunması Protein Expression and Purification Why? Obtain pure (clean) protein "Don't waste clean thinking on dirty enzymes“ - Arthur Kornberg Powerful experimental tool Simplifies the system in which you are asking a question Confirmation of a hypothesis that is developed in a more complex system 16 Protein Expression Why over-express the protein? Make large quantities to facilitate purification/study Analyze biochemical properties Perform structural analyses Crystallization NMR Identify protein interactions Make Antibodies Expression systems E. coli Yeast For bacterial or eukaryotic proteins Large amounts of protein Insect cells Prokaryotic expression workhorse Post-translational modifications In-vitro systems wheat germ, rabbit reticulocyte 17 Purification strategies Exploiting protein chemistry Size/Mass Charge Hydrophobicity Antibody affinity Protein Tags Often used in combination Separation processes that can be used to fractionate proteins Separation Process Basis of Separation Precipitation ammonium sulfate polyethyleneimine (PEI) isoelectric solubility charge, size solubility, pI Chromatography gel filtration (SEC) size, shape ion exchange (IEX) charge, charge distribution hydrophobic interaction(HIC) hydrophobicity DNA affinity DNA binding site immunoaffinity (IAC) specific epitope chromatofocusing pI Electrophoresis gel electrophoresis (PAGE) charge, size, shape isoelectric focusing (IEF) pI Centrifugation Ultrafiltration sucrose gradient size shape, density ultrafiltration (UF) size, shape 18 Size Exclusion Chromatography Separation based on size of protein Ion exchange 19 HPLC High performance (pressure) liquid chromatography Sample is passed over column of varying hydrophobic nature- more hydrophobic particles bind tighter and elutes later. Eluate is analyzed by a detector UV, refractive index, fluorescence Can be combined with mass spec (LC/MS) HPLC 20 Affinity/Ab columns Purify tagged proteins Interaction between two molecules Solid phase- immobilized on column Mobile phase- binds while passing over column Buffer conditions regulate binding & dissociation pH, ionic strength, competing Ab affinity column 21 Tagging the protein Clone gene in frame with a unique protein sequence or “tag” Advantages Purification • Use tag to selectively remove protein from a complex sample Protein visualization/tracking • Fluorescent protein tags, labeled antibodies Protein Tags His small 6 HIS residues bind to nickel or cobalt columns GST Binds to glutathione resin/beads S-Tag, C-myc, HA, flag Antibody affinity columns 22 Protein Tags 6xHistidine binds metal chelating resinCu2+, Ni2+, Co 2+ Commercially available protein purification kits •GST•Bind™ Purification Kits •His•Bind® Purification Kits •Magnetight™ Oligo d(T) Beads •MagPrep® Streptavidin Beads •Protein A and Protein G Plus Agaroses •S•Tag™ Purification Kits •Streptavidin Agarose •T7•Tag™ Affinity Purification Kit •ProteoSpin™ CBED (Concentration, Buffer Exchange and Desalting) Maxi Kit — Effectively desalts and concentrates up to 8 mg of protein with an efficient, easyto-use protocol.(Norgen Biotek Corporation) •ProteoSpin™ Detergent Clean-up Micro Kit — Provides a fast and effective procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40 and Tween 20. (http://www.emdbiosciences.com) 23 İnce Tabaka Kromatografisi (TLC) •Proteinlerin çözünme yeteneklerine göre ayrışım yapılır. •Katı bir yüzeye (selüloz) emdirilen örnek çözücü yüzey içine yerleştirilir. Çözücü solüsyonun yüzeyde ilerlemesiyle, örnekteki proteinler çözünme yeteneklerine göre ayrılırlar. What can you do with purified proteins? Biochemical and functional characterization Structural analyses informs function DNA binding, enzyme activity, stability, etc., NMR, crystallography, circular dichroism Study protein-protein or protein-DNA interactions Develop antibodies, therapeutics 24
