Datum Přednáška Seminář 24.9. Úvod do studia, Koncepce přípravy rekombinantních proteinů, Základní analytické pojmy x 1.10. Separační a Chromatografické metody, Ostatní separační metody: Fokusace, Ultracentrifugace, Dialýza Úloha 1_TLC 8.10. Spektroskopické techniky I : UV-Vis, Fluorescence, FRET x 15.10. Spektroskopické techniky II: CD, vibrační spektroskopie: IC, Raman, X-Ray, QD Úloha 2_CD 22.10. NMR a EPR x 29.10. Optická a elektronová mikroskopie (SEM,TEM), AFM, Fluorescenční mikroskopie, Konfokální mikroskopie, Klasická mikroskopie x 5.11. MS, LC-MS, Aplikace MS Úloha 3_MALDI-TOF MS 12.11. Úvod do elektrochemie (pH, pKa, Nernstova rovnice, Voltametrie, Potenciometrie, Amperometrie, Impedanční a Pulzní Voltametrie + jejich aplikace) x 19.11. Elektroforéza - Gelová ELFO, Kapilární ELFO, Aplikace Úloha 4_SDS-PAGE 26.11. Bio-elektrochemie - Rozptyl, X-Ray, Biosenzory, Aplikace, SPR. x 3.12. Strukturní biochemie - Techniky pro určování 3D struktur proteinu (X-ray, NMR, cryoEM, simulované žíhání) Exkurze 1, pavilon C4 (Laboratoř přípravy proteinu a NMR park) 10.12. Biointerakce - Metody pro určování interakcí a oligomerních stavů - BioKalorimetrie (Termostabilita, ITC a DSC + alternativní interakce/hydrodynamické techniky (osmometrie, centrifugace, Svedberg). Exkurze 2, pavilon C4 (ITC, DLS, nanoDSF...) 17.12. Metody lékařské diagnostiky Exkurze 3, pavilon C14 (MS, MALDI-TOF MS, ESI-Orbitrap, DROBECEK (MS)) Sylabus – Přednášky a semináře C5855 a C5856, podzim 2024 Exprese a purifikace proteinů Jozef Hritz (jozef.hritz@ceitec.muni.cz) Central Dogma of Molecular Biology • Biology Pictures: Genetic Code (Circular) Team:IISc-Bangalore/Design - 2017.igem.org Codon Usage 15.2: The Genetic Code - The Central Dogma- DNA Encodes RNA and RNA ... Codon optimized sequence can be often achieved by synthetic gene synthesis - > particularly useful for the projects where the large expressions are needed, e.g. structural biology. Or isotopic labeling… Different codon usage e.g. BL21-Codon plus-RIL Often cleavage sites are considered •Thrombin (optimal pH ~8.0) •Pro-Arg/Gly •Pro-Lys/Leu •Ala-Arg/Gly •Gly-Lys/Ala •Ile-Arg/Ser •Leu-Arg/Ala •Ile-Arg/Ile • •TEV protease •Glu-Asn-Leu-Tyr-Phe-Gln/Ser •pH 5.5 –8.5 Phusion Tags •a) short peptides [ex. (His)n, (Asp)n, (Arg)n ... ]. •b) protein domains, entire proteins • [ex. MBP, GST, thioredoxin …]. • • • •Purpose •Increasing the yield of recombinant proteins – Fusion of the N-terminus of the target protein to the C-terminus of a highly expressed fusion partner results in high level expression of the target protein. •Enhancing the solubility of recombinant proteins – Fusion of the N-terminus of the target protein to the C-terminus of a soluble fusion partner often improves the solubility of the target protein. •Facilitating the purification of recombinant proteins – Simple purification schemes have been developed for proteins used at either terminus which bind specifically to affinity resins. Once there is clear idea of DNA sequence Gene amplification by PCR insertion into the vector by e.g. restriction endonucleases Graphical user interface, diagram, application Description automatically generated A picture containing circle, graphics, art Description automatically generated •circular E.coli plasmids (in addition to E.coli genome) – why antibiotic resistance is needed? •Lac operator • • • lac operon, lac promoter, lac operator, lactose metabolism PET vector, lacI gene, LacO, T7 Promoter The diagram of glycolysis/gluconeogenesis partial pathway presents the ... target gene pET plasmids 1.3.2.1. Exact molecular mass 1.3.2.2. Isoelectric point 1.3.2. Information available from the amino acid sequence of a protein 1.3. The primary structure of proteins http://www.expasy.ch/tools/pi_tool.html C:\Users\Admin\Desktop\Prednaska\Lecture1\ch01f10.jpg 9 1.3.2.3. Absorption coefficient 1.3.2.4. Hydrofobicity 1.2.2. Clasification of the amino acids in terms of polarity Non-polar side chain Ala, Gly, Ile, Leu, Met, Phe, Pro, Trp, Val Polar, uncharged side chain Asn, Cys, Gln, Ser, Thr, Tyr Polar charged side chain Arg, Asp, Glu, His, Lys 1.2.1. The variety of amino acids 1.2. The amino acids 10 1.2.3.2. Ionization 1.2. The amino acids 1.2.3. General properties of the amino acids 11 http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/proteins.htm Transformace Colony selection IPTG Exprese IPTG What kind of growing media? Purifikace •Affinity chromatography - Immobilized metal ion affinity chromatography (IMAC) • • • • • • • • •Ion-exchange chromatography •Optimal binding of recombinant protein with metal ion is achieved at pH 7–8. •Buffers with a high salt concentration (0.5–1 M NaCl) reduce nonspecific electrostatic interaction. •Elution of contaminating proteins can be achieved by lowering the pH or using low concentrations of imidazole. •Elution of tagged protein is achieved at high imidazole concentrations (0–0.5 M), by strongly decreasing the pH, or by using EDTA. Size-exclusion chromatography (Gel filtration) •porous beads •Size-exclusion chromatography separates proteins • on the basis of size Protein Purity •SDS-PAGE • • • • • •Mass spectrometry (MS)-intact analysis, e.g. by MALDI-TOF MS Another properties to check: •secondary structure (e.g. CD) •thermostability (typically in terms of Tm) •oligomeric state (e.g. DLS, AUC) What is molecular weight of 14-3-3zeta homodimer? •Viac detailne a hlavne prakticke informacie na danu temu - v ramci predmetu: C8980 Příprava a charakterizace proteinů I - Exprese a purifikace