The characterization of Amino Acids and the Purification of Proteins

1. The characterization of Amino Acids and the Purification of Proteins Shawndell Powell High School for Medical Science Bronx, NY, 10457 Dr. Jeffrey O. Boles Chemistry Department Tennessee Technological University Cookeville, TN, 38505

2. Amino Acids • A class of organic compounds that contain both the amino and carboxyl groups. • Of these acids, 20 serve as the building blocks of proteins . • Known as the standard, or alpha, amino acids, they comprise serine and tryptophan which are 2 of the 20 that are constructed according to a general formula: R O H2N C C OH H

3. Proteins • A large number of organic compounds that make up living organisms and are essential to their functioning. • Whether found in humans or in single-celled bacteria, proteins are composed of units of about 20 different amino acids

4. Goal: To make selenatryptophan more commercially available Objective: Run a TLC (Thin Layer Chromatography) Purify Proteins

5. the enzyme that were over expressed in bacteria and then purified using medium pressure liquid chromatography commercially available buffer to control pH Se-Indole analog substrate synthesized at Los Alamos National Lab to make with 90% yield RxN centraprep to remove enzyme selenatryptophan product analysis by TLC & NMR better yield reproducibility

6. Running a TLC • Dissolve 10mg (0.01g) into 2mL of dH2O • Vortex until in solution

7. Running a TLC • Spot sample on a TLC plate • Set in TLC solution for about 10-15mins.

8. Running a TLC • Look at it under a light • Circle samples movement

9. Running a TLC • Set it in a jar of iodine or spray it with ninhydrant • Then dry it with a plow dryer

10. Results

11. Purifying Proteins Wash Mode: • Turn on recorder, detector, gradifrac, and P-50 pump. Put on recorder pens • In “wash” mode with the P-1 pump, pass H2O of 10% EtOH through P-1 out port 4 to waste, then turn P-1 off • Change to “inject” mode, turn P-1 on and pass 2-3mL of H20 or 10% EtOH through the column • Turn P-1 off, switch to Buffer A, turn on P-1 on for 2-3mins.

12. P-50 Preparation: • Drop A & B line of P-50 into Buffer A and Buffer B, change to “wash” mode • Run method 0 on gradifrac

13. Loading CFE (Cell Free Extract): • Change to “inject” mode • Put P-1 line in Buffer A and pump 2mls • Turn off P-1 pump • Go into manual mode on gradifrac and set it to 0% B, 2ml/min, 8ml fractions, and put line 6 in Buffer A after line 6 spends 2mins in waste beaker

14. Loading CFE (Cell Free Extract): • Pump Buffer A through P-1 to equilibrate the column. Turn P-1 off and press “pause” on gradifrac • Put P-1 line in CFE, turn on P-1, press “continue” on gradifrac and load the CFE, turn off P-1 and switch line back to buffer A • Continue with Buffer A through P-1 for 10-30mins.(until the recorder approaches the base line • Press “end” on gradifrac, turn off P-1 • Change valve to “load” mode and run method

15. Stripping Chelating Sepharose: • Push “end” on gradifrac • Set valve to “inject” mode • Pump P-1 line in strip buffer and turn on P-1 on • Pump until column is white • Turn off P-1 and change line to 10% EtOH • Run 10% EtOH through column for 10mins

16. After finishing your chromatography: • Push “end” on gradifrac • Change valve to “inject” mode, put P-1 line in 10% EtOH and turn P-1 on for 10mins. Turn P-1 pump • Change valve to “wash” mode, drop P-50 A & B lines into 10% EtOH and run method 9 on gradifrac • Turn all power off.

17. Acknowledgements • Dr. Boles • Jeffrey • Dr. Dan • Dr. Sat • Harlem Children Society • Chemistry Dept. at TTU