Proteins as Products PDF

Introduction to Proteins as Products TOPIC 3  Why proteins are important? ❖ They serve as catalysts that maintain metabolic processes in the cell. ❖ They serve as structural elements both within and outside the cell. ❖ They are receptors that convey information about the extra...

Introduction to Proteins as Products TOPIC 3  Why proteins are important? ❖ They serve as catalysts that maintain metabolic processes in the cell. ❖ They serve as structural elements both within and outside the cell. ❖ They are receptors that convey information about the extracellular environment to the cell. ❖ They serve as intracellular signaling components that mediate the effects of receptors. ❖ They are key components of the machinery that determines which genes are expressed and whether mRNAs are translated into proteins. ❖ They are involved in manipulation of DNA and RNA through processes such as: DNA replication, DNA recombination, RNA splicing or editing. Review of the Basics ⚫ Made up of amino acids ⚫ Functions: ⚫ Regulatory role ⚫ Structural support ⚫ Transport ⚫ There are literally thousands of functions and we do not yet understand all of them! ⚫ In order to understand their functions we have to understand their structure Protein Structure ⚫ Polymers of small units (amino acids) ⚫ Proteins do NOT have a uniform structure ⚫ Due to 20 different amino acids available ⚫ The chemical and physical properties are different among the different amino acids ⚫ Protein sequence reported by Frederick Sanger in 1953. ⚫ Protein folding determines structure Protein Structures ⚫ Levels of Organization ⚫ Primary (the AA sequence of its polypeptide chain) ⚫ Secondary (H bonding between peptide bonds) ⚫ Tertiary (covalent, ionic, H bonding, hydrophobic) ⚫ Quaternary (involves more than one subunit) Examples of Purified Proteins ⚫ Enzymes ⚫ Amylases, proteases, lipases (google the company, Genzyme-how many of the enzymes does this company make?)http://www.genzyme.com/business/biz_ho me.asp ⚫ http://www.genzymediagnostics.com/ ⚫ Hormones ⚫ Antibodies ⚫ What was the first recombinant protein to be mass produced? ⚫ Cerezyme More Examples of products: ⚫ Food Processing (the creamy in ice cream) ⚫ Textiles and Leather Goods ⚫ Medical and Pharmaceuticals ⚫ Detergents (enzymes) ⚫ Paper Manufacturing and Recycling ⚫ Bioremediation: Treating Pollution with Proteins (metallothioneins) Protein Secretion Pathway Wang et al. 2020 Protein folding ⚫ Folding describes how different strands of amino acids take their shape. ⚫ If a protein folds incorrectly, not only will the desired function of the protein be lost but the resulting mis-folded protein can be detrimental. ⚫ Exm: SCA, Alzheimer’s disease, mad cow disease Protein folding Glycosylation ⚫ In glycosylation, carbohydrate units (sugar molecules) are added to specific locations on proteins. ⚫ This change can have a significant effect on a protein’s activity in an organism. ⚫ it can increase solubility and orient proteins into membranes and ⚫ extend the active life of a molecule Glycosylation Turning Proteins into Products ⚫ Identifying proteins and their function is only half the battle. ⚫ Once identified, proteins typically need to be grown and then purified and processed into usable, salable products. ⚫ Levels of Product Purity (least to most pure) ⚫ Research grade ⚫ Diagnostic grade ⚫ Pharmaceutical grade (low to high dose)  The Making of Protein Therapies https://www.youtube.com/watch?v=a6Rqzq_cJaY Protein Production Upstream Processing: the actual expression of the protein in the cell ⚫ Microorganisms- cheap, well understood, grow rapidly, produce large amounts, clone in as cDNA, fusion gene (fusion protein), inclusion bodies, no glycosylation ⚫ Fungi – can do some posttranslational modifications ⚫ Plants- 85% of current drugs from plants; rapid growth, cheap, proteins not expressed properly ⚫ Mammalian Cell Systems – finicky, grow slowly, and expensive, BUT processes human proteins correctly ⚫ Whole-animal –transgenic (goats making spider silk) ⚫ Insect systems – baculoviruses are used as vector to insert genes into insect cells Recombinant Protein and Its Expression Systems https://www.youtube.com/watch?v=r7RE2aAqPs4 Downstream Processing: the protein is separated from other parts of the cell and then isolated from other proteins ⚫ Step 1: Preparing the Protein Extract for Purification ⚫ If intracellular, lyse the cells ⚫ Detergents or organic solvent can be used for lipid membrane bound proteins ⚫ Step 2: Stabilizing the Proteins in Solution ⚫ Temperature, decrease protease activity and denaturing activity, maintain biological activity ⚫ Step 3: Separating the Components in the Extract ⚫ Utilize the chemical and physical properties of proteins to separate them Protein Purification by Size Exclusion Chromatography Protein Purification by Ion-Exchange Chromatography Protein Purification by Size Exclusion Chromatography  Verification ⚫ SDS-PAGE File:SDS-PAGE Electrophoresis.png Compare protein size to set of sizing standards run SDS Page ⚫ Electrophoresis process used for proteins: can determine molecular weight of a protein ⚫ The SDS (sodium dodecyl sulfate) helps to unfold protein Process ⚫ Unfolds the protein to make it linear ⚫ Separates the protein and subunits by molecular weight ⚫ Coats the protein with negative charge (run like gel electrophoresis)