1. Identifying a Potential Biotechnology Product Chapter 6

2. Objectives Describe the CPDP process. Describe the CPDP process. List and describe the different assays that can be performed on proteins. List and describe the different assays that can be performed on proteins. Describe the processes that can used to isolate molecules of interest from plant cells. Describe the processes that can used to isolate molecules of interest from plant cells. Describe how plant proteins of interest can be mass produced by cells. Describe how plant proteins of interest can be mass produced by cells.

3. Is it worth pursing a product or not? Biotechnology products can come from a variety of sources. Biotechnology products can come from a variety of sources. Decision needs to be made as to whether it is worth pursuing the development and sale of a product. Decision needs to be made as to whether it is worth pursuing the development and sale of a product. Many companies use a CPDP strategy. Many companies use a CPDP strategy. Comprehensive Product Development Plan Comprehensive Product Development Plan

4. Is it worth pursing a product or not? CPDP 1. Does the product meet a critical need? Who will use the product? 2. Is the market large enough to produce sufficient sales? How many customers are there? 3. Does the preliminary data support that the product will work? Will it do what the company claims? 4. Can patent protection be secured? Can the company prevent other companies from producing it? 5. Can the company make a profit on the product? How much will it cost to make? How much can it be sold for? I.e. common cold vaccine I.e. common cold vaccine Amylase Amylase

5. Verifying what you have. A majority of the biotechnology products produced are proteins. A majority of the biotechnology products produced are proteins. Must be able to determine: Must be able to determine: Is product present? Is product present? What is the overall activity of the product? What is the overall activity of the product? Concentration of product. Concentration of product. Difficulty in working with proteins is that they are colorless and nonvisible to the naked eye. Difficulty in working with proteins is that they are colorless and nonvisible to the naked eye.

6. Assays (Tests) 1. Activity assays 1. Activity assays 2. Concentration assays 2. Concentration assays Bradford protein reagent or bicinchoninic acid (BCA) Bradford protein reagent or bicinchoninic acid (BCA) Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay (ELISA)

7. Verifying what you have. Generally assays, meaning test, are done when working with proteins. Generally assays, meaning test, are done when working with proteins. Activity assay Activity assay Indicate presence Indicate presence Indicate whether protein is working or not Indicate whether protein is working or not Lighter color…..more active amylase

8. Verifying what you have. Concentration assay Concentration assay Amount/unit volume of a protein Amount/unit volume of a protein By eye or spectrophotometer By eye or spectrophotometer Bradford protein reagent or bicinchoninic acid (BCA) Bradford protein reagent or bicinchoninic acid (BCA) Nonspecific in its action Nonspecific in its action ELISA ELISA Enzyme-linked immunosorbent assay Enzyme-linked immunosorbent assay Very specific Very specific Unitize antigen-antibody specifity Unitize antigen-antibody specifity An enzyme attached to an antibody binds to a specific protein (antigen) An enzyme attached to an antibody binds to a specific protein (antigen) Causes a color change that can be meas. When specific substrate is added Causes a color change that can be meas. When specific substrate is added Decreasing amount of protein…..indicated by less blue color in tube Negative 0.01, 0.1, 1mg/mL The darker the color…..higher conc. Of protein

9. Verifying what you have. Dealing with pharmaceuticals Dealing with pharmaceuticals The stringency of verifying what you have, that it is safe, and that it works are much higher. The stringency of verifying what you have, that it is safe, and that it works are much higher. In addition to activity and conc. In addition to activity and conc. PK assay PK assay Pharmacokinetic (amount of activity of protein in humans and other test organisms) Pharmacokinetic (amount of activity of protein in humans and other test organisms) PD assay PD assay Pharmacodynamic (length of activity) Pharmacodynamic (length of activity) Additional assays developed and carried out to show potency, toxicology, and stability of drug. Additional assays developed and carried out to show potency, toxicology, and stability of drug. Potency: how dosage of a drug affects its activity and how long stays in body Potency: how dosage of a drug affects its activity and how long stays in body Toxicology: what quantities of drug toxic to cells, tissues, and model organism Toxicology: what quantities of drug toxic to cells, tissues, and model organism Stability: shelf life, and storage conditions Stability: shelf life, and storage conditions Assay Services and Quality Control Department. Assay Services and Quality Control Department.

10. Focus on Plant Proteins as Possible Products Extracting Protein Molecules from Cells Extracting Protein Molecules from Cells Most molecules of interest are found within the cell. Most molecules of interest are found within the cell. Problems encountered when attempting to isolate RNA, DNA, or proteins from plant cells. Problems encountered when attempting to isolate RNA, DNA, or proteins from plant cells. Plant material is dense and can be quiet woody. Plant material is dense and can be quiet woody. Cells that make up plants have cell walls that are very thick. Cells that make up plants have cell walls that are very thick. Plant material broken into smaller components to increase surface area for solvents to work on. Plant material broken into smaller components to increase surface area for solvents to work on.

11. Focus on Plant Proteins as Possible Products Removing or Weakening Cell Walls Removing or Weakening Cell Walls Enzymatic removal of cell wall Enzymatic removal of cell wall Major components of cell wall are cellulose and pectin. Major components of cell wall are cellulose and pectin. Cellulase and pectinase enzymes degrade cellulose and pectin. Cellulase and pectinase enzymes degrade cellulose and pectin. Once cell wall removed protoplast forms. Once cell wall removed protoplast forms. Plant cell with only a plasma (cell) membrane. Plant cell with only a plasma (cell) membrane. Homogenization of plant cells Homogenization of plant cells Crushing action that results in release of intracellular contents. Crushing action that results in release of intracellular contents. I.e. Tissuemizer I.e. Tissuemizer Works like blender Works like blender Grinds tissues without causing foam Grinds tissues without causing foam

12. Focus on Plant Proteins as Possible Products Removing or Weakening Cell Walls Removing or Weakening Cell Walls Freeze facture Freeze facture Cells flash frozen with liquid nitrogen or dry ice Cells flash frozen with liquid nitrogen or dry ice Crushed using a mortar and pestle Crushed using a mortar and pestle Results in maceration of cells Results in maceration of cells Isolating Proteins Isolating Proteins Cell contents placed in a neutral solution and proteins extracted. Cell contents placed in a neutral solution and proteins extracted. i.e. potassium phosphate buffer i.e. potassium phosphate buffer Other extraction methods that can be used are acetone or ammonium sulfate. Other extraction methods that can be used are acetone or ammonium sulfate. Once protein(s) isolated assays are generally run. Once protein(s) isolated assays are generally run.

13. Focus on Plant Proteins as Possible Products Once a particular protein has been identified a possible way to make more of it is to produce rDNA. Once a particular protein has been identified a possible way to make more of it is to produce rDNA. Cells can be transformed with the rDNA and begin the process of making the protein. Cells can be transformed with the rDNA and begin the process of making the protein.

14. Homework Review questions page 168 Review questions page 168 Questions 1, 2, 3, and 4 Questions 1, 2, 3, and 4 Think like biotech page 180 Think like biotech page 180 Questions 3, 5, 6, 7, and 9 Questions 3, 5, 6, 7, and 9 This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity. This product was funded by a grant awarded under the President’s High Growth Job Training Initiative, as implemented by the U.S. Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual organizational, non-commercial use only.