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Chapter 4 Proteins as Products Proteins as Biotech Products Enzymes – speed up chemical reactions • Synthesis – combines small molecules to make larger molecules • ATP synthetase, peptidyl transferase, polymerase • Depolymerization – breaks down large molecules • Amylase, lipase, protease Hormones – carry chemical messages Antibodies – part of immune response Proteins as Biotech Products Therapeutic Protein • used to treat a disease that is caused by a gene that fails to produce a necessary protein or that produces a dysfunctional protein Proteins as Biotech Products Food processing – the creamy in ice cream Textile and leather goods – bio-bleaches Detergents – enzymes to dissolve stains Paper manufacturing and recycling – reduce negative environmental impacts Adhesives – barnacles and mussels Bioremediation – proteins used to clean up harmful waste Central Dogma DNA codes for RNA which codes for proteins. Translation A protein is a string of amino acids held together by peptide bonds and do most of the work in a cell Translation Translation Protein Structure Once the amino acid chain is released from the ribosome, a number of modifications are made in order for the protein to perform it’s intended function. The protein must fold into it’s appropriate 3-dimensional shape. Protein Structure Proper folding of the protein is essential for it’s activity because it must bind it’s substrate to perform it’s job. Protein Structure Primary – Peptide bonds in a chain of amino acids Secondary – Hydrogen bonding between amino acids forms alphahelices and beta-sheets Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and betasheets Quaternary – aggregation of multiple polypeptide chains Protein Structure Glycosylation • Carbohydrate units added to protein • Increases solubility, orients protein in membrane, extends life of protein • Occurs in the golgi Protein Structure 5’-GATCTGAATCGCTATGGC-3’ Coding: Template: 3’-CTAGACTTAGCGATACCG-5’ mRNA: mRNA 5’-GAUCUGAAUCGCUAUGGC-3’ tRNA: CUAGACUUAGCGAUACCG amino acid: Asp, Leu, Asn, Arg, Tyr, Gly Protein Structure DNA codes for proteins that confer traits Protein Engineering Directed Molecular Evolution • Introducing specific, predefined alterations in the DNA sequence. Protein Production Steps in bioprocessing Protein Expression Bacteria Advantages Cheap and easy to grow Biology is well-defined High yield of recombinant proteins in culture Disadvantages • Many proteins become insoluble in inclusion bodies • Most if not all post-translational modifications are not added Protein Expression Fungi Advantages • Grown in simple, inexpensive media • Secrete many proteins into the media • Capable of many post-translational modifications Disadvantages • Recombinant proteins usually expressed at low levels • Some post-translational modifications differ significantly Protein Expression Plants Advantages • Rapid growth and reproductive rates • Perform most post-translational modifications • Transgenic plants can be self-fertilized Disadvantages • Not all mammalian proteins are expressed in plants • Plant cells have a tough cell wall • Some plants produce proteins in their green leaf tissues Protein Expression Mammalian Cell Culture Advantages • Protein-folding and post-translational modification • Powerful promoters to regulate protein expression • High expression levels Disadvantages • Complex and expensive nutritional requirements • Slow growing Protein Extraction The target protein must be separated from the complex mixture of biological molecules Protein Extraction Isolated proteins must be stabilized Very sensitive to changes in temperature Proteases that could digest the target protein are a threat Protein folding is dependent on the pH of the environment Protein Purification Chromatography A method to separate proteins by size, charge, or chemical properties as they pass through a column of resin beads Chromatography Animation Protein Purification Chromatography Resin/matrix – solid particles in the column Sample – protein mixture that is loaded on the column Elution – liquid that passes through the column and is collected in fractions Protein Purification Size Exclusion Chromatography Separates proteins based on size Small molecules get caught in the beads Larger molecules pass quickly around the beads and elute first Protein Purification Ion Exchange Chromatography Separates molecules based on ionic charge Proteins are eluted by increasing the concentration of a salt buffer Proteins with the weakest charge are eluted first Protein Purification Hydrophobic Interaction Chromatography Separates proteins based on repulsion to water Proteins are eluted by decreasing the salt concentration of the buffer The least hydrophobic proteins are eluted first Protein Purification Affinity Chromatography Separates proteins based on molecular conformation Matrix is made of a ligand specific for the desired protein The protein is cleaved from the matrix using a site-specific protease Protein Purification High Performance Liquid Chromatography (HPLC) Applies high pressure to drive sample through the column faster Protein Verification SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) Separates proteins in an electrical field based on molecular size Protein Verification Sodium Dodecyl Sulfate (SDS) A detergent that denatures the secondary and tertiary structure of the protein Coats the protein with negative charges Add SDS Protein Verification Polyacrylamide Gel Electrophoresis (PAGE) Much tighter gel matrix than agarose, which makes polyacrylamide ideal for separating proteins Protein Verification SDS-PAGE to test for purity Protein Verification SDS-PAGE Animation Application Recombinant human insulin Preserving Proteins Lyophilization (freeze drying) • Placed under vacuum to hasten evaporation of water • Containers are sealed after water is removed Scale-up of Protein Purification R&D works on small scale Large production demands protocols to scale-up bioreactors • If FDA approval has been gained for small-scale, cannot change the parameters when scaled up Postpurification Analysis Protein Sequencing • Determining the order of amino acids X-ray Crystallography • Determining tertiary and quaternary structure of protein Proteomics Proteomes are compared under healthy and diseased states • The variations of protein expression are then correlated to onset or progression of a specific disease Protein Microarrays • Identifies protein interactions