Download presentation
Presentation is loading. Please wait.
1
Chapter 4 Proteins as Products
2
Chapter Contents 4.1 Introduction to Proteins as Biotech Products
Proteins as Biotechnology Products Protein Structures Protein Production Protein Purification Methods Verification Preserving Proteins Scale-Up of Protein Purification Postpurification Analysis Methods 4.10 Proteomics
3
4.1 Introduction to Proteins as Biotech Products
Proteins – large molecules that are required for the structure, function, and regulation of living cells 2000 NIH launched Protein Structure Initiative Effort to identify the structure of human proteins
4
4.2 Proteins as Biotechnology Products
Use of proteins in manufacturing is a time-tested technology Beer brewing and winemaking Cheese making Recombinant DNA technology made it possible to produce specific proteins on demand Enzymes – proteins that speed up chemical reactions Hormones Antibodies
5
4.2 Proteins as Biotechnology Products
Making a Biotech Drug Produced through microbial fermentation or mammalian cell culture Complicated and time-consuming process Must strictly comply with FDA regulations at all stages of the procedure
6
4.2 Proteins as Biotechnology Products
Applications of Proteins in Industry Medical applications Food processing Textiles and leather goods Detergents Paper manufacturing and recycling Adhesives: natural glues Bioremediation: treating pollution with proteins
7
4.3 Protein Structures Proteins
Are complex molecules built of chains of amino acids Have electrical charge that causes them to interact with other atoms and molecules Hydrophilic – water loving Hydrophobic – water hating
8
4.3 Protein Structures Structural Arrangement – four levels
Primary structure is the sequence in which amino acids are linked together Secondary structure occurs when chains of amino acids fold or twist at specific points Alpha helices and beta sheets Tertiary structures are formed when secondary structures combine and are bound together Quaternary structures are unique, globular, three-dimensional complexes built of several polypeptides
9
4.3 Protein Structures
10
4.3 Protein Structures Protein Folding
The structure and function of a protein depends on protein folding If protein is folded incorrectly, desired function of a protein is lost and a misfolded protein can be detrimental two regular structures were described Alpha helices and beta sheets Structures are fragile; hydrogen bonds are easily broken
11
4.3 Protein Structures Glycosylation – post-translational modification wherein carbohydrate units are added to specific locations on proteins More than 100 post-translational modifications occur
12
4.3 Protein Structures Protein Engineering
Introducing specific, predefined alterations in the amino acid sequence through a process known as directed molecular evolution technology Creating entirely new protein molecules
13
4.4 Protein Production Proteins are valuable
Proteins are complex and fragile products Production of proteins is a long and painstaking process Upstream processing includes the actual expression of the protein in the cell Downstream processing involves purification of the protein and verification of the function; a stable means of preserving the protein is also required
14
4.4 Protein Production Protein Expression: The First Phase in Protein Processing Selecting the cell to be used as a protein source Microorganisms Fungi Plants Mammalian cell systems Whole-animal production systems Insect systems
15
4.5 Protein Purification Methods
Protein Must Be Harvested Entire cell is harvested if protein is intracellular Requires cell lysis to release the protein Releases the entire contents of the cell Culture medium is collected if the protein is extracellular
16
4.5 Protein Purification Methods
Separating the Components in the Extract Similarities between proteins allow the separation of proteins from non-protein material Protein precipitation – salts cause proteins to settle out of solution Filtration (size-based) separation methods Centrifugation Membrane filtration Microfiltration Ultrafiltration
17
4.5 Protein Purification Methods
Separating the Components in the Extract Diafiltration and dialysis rely on the chemical concept of equilibrium
18
4.5 Protein Purification Methods
Separating the Components in the Extract Differences in proteins allows the separation of the target protein from other proteins Chromatography – allows the sorting of proteins based on size or by how they cling to or dissolve in various substances
19
4.5 Protein Purification Methods
Separating the Components in the Extract Chromatography Size exclusion chromatography (SEC) – uses gel beads with pores Larger proteins move quickly around the beads and smaller proteins slip through the pores and therefore move more slowly through the beads
20
4.5 Protein Purification Methods
21
4.5 Protein Purification Methods
Separating the Components in the Extract Chromatography Ion exchange chromatography – relies on the charge of the protein Resin is charged Opposite charged proteins will stick to resin beads Can be eluted by changing the charge with salts of increasing concentration
22
4.5 Protein Purification Methods
23
4.5 Protein Purification Methods
Separating the Components in the Extract Chromatography Affinity chromatography relies on the ability of proteins to bind specifically and reversibly to uniquely shaped compounds called ligands
24
4.5 Protein Purification Methods
Separating the Components in the Extract Chromatography Hydrophobic interaction chromatography (HIC) sorts proteins on the basis of their repulsion of water
25
4.5 Protein Purification Methods
26
4.5 Protein Purification Methods
Separating the Components in the Extract Iso-electric focusing used in QC to identify two similar proteins that are difficult to separate by any other means Each protein has a specific number of charged amino acids on its surface in specific places Creates a unique electric signature known as its iso-electric point (IEP) where charges on the protein match the pH of the solution
27
4.5 Protein Purification Methods
Separating the Components in the Extract Analytic methods High-Performance liquid chromatography (HPLC) – uses high pressure to force the extract through the column in a shorter time Mass spectrometry (mass spec) – highly sensitive method used to detect trace elements Used to indicate the size and identity of most protein fragments
28
4.6 Verification The presence and concentration of the protein of interest must be verified at each step of the purification process SDS-PAGE (polyacrylamide gel electrophoresis) Western blotting ELISA
29
4.7 Preserving Proteins Lyophilization (freeze-drying)
Protein, usually a liquid product, is first frozen A vacuum is used to hasten the evaporation of water from the fluid Will maintain protein structure and can be stored at room temperature for long periods of time
30
4.8 Scale-Up of Protein Purification
Protocols are usually designed in the laboratory on a small scale Must be scaled up for production Process is approved by FDA so must make sure laboratory procedures can be scaled up
31
4.9 Postpurification Analysis Methods
Protein Sequencing Must determine the primary structure, the sequence of amino acids X-ray Crystallography Used to determine the complex tertiary and quaternary structures
32
4.10 Proteomics A new scientific discipline dedicated to understanding the complex relationship of disease and protein expression Uses protein microarrays to test variation in protein expression between healthy and disease states
Similar presentations
