Protein Purification from Corn Germ

Danielle McConnell

Department of Chemical Engineering

Iowa State University

Possible Pathways for Protein Purification

Ultrafiltration with a 10 kD membrane

Ultrafiltration witha 1 kD membrane

Cation Exchange

Purified form of Protein

Cation Exchange

Heat Treatment at 10 minutes

Corn Germ

Extraction at pH 4

Ultrafiltration with

Purified form of Protein

Ultrafiltration with a 1 kD membrane

a 10 kD membrane

Why extract Protein X from Transgenic Corn?

• Could be a good alternative for a low calorie sweetener

• 9500 times sweeter than sucrose on a molecular basis

• Good extraction attributes (heat-stable and low molecular weight)

• Transgenic corn is economically feasible, easy to scale up, and is highly available

Extraction (part 1)

pH determination

• Isoelectric point of Protein is 5.72

• Tested pHs 4,5,6, and 7

• Total Protein Assays

• pH 4 showed the least amount of total protein extracted

pH Buffer Total Protein

(g/ml)4 20 mM sodium

acetate554

5 20 mM sodiumacetate

2337

6 20 mM Tris-HCl 49107 20 mM sodium

phosphate8234

Extraction (part 2)

Determination of Heating Time

• At 80 ° C Protein is thermo-stable• Total Protein Assays of precipitate at different times• At ten minutes the total protein is the smallest

Heating Time Total Protein(g/ml)

0 (original) 540.710 min 443.130 min 451.1

Cation Exchange Chromatography

• Proteins bind to the negatively charged clusters by electrostatic forces

• A SP-Sepharose column was used to run the ion exchange

• According to the chromatogram (see next panel), peak 6 or fraction #38 displays the protein rich sample

Cation Exchange Setup shown above

0

50

100% Buffer B

2 3

4

6

8

9

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.00

0.50

1.00

1.50

2.00

2.50

00:00:00 02:00:00 04:00:00 06:00:00 08:00:00 10:00:00

Fractions

Hr:Min:Sec mS/cmAU

Cation Exchange Chromatogram

Conductivity vs. Time (fraction # at top of graph)

Ultrafiltration

• Used a used large and small molecular weight cutoff membranes to filter the extract

• Protein should be left in the retentate of the small MW cutoff membrane

• Total Protein Assays gave a preliminary evaluation of what was left in the retentate

Original ExtractTotal Protein=539.1 ug/ml

Heated ExtractTotal Protein=454.3 ug/ml

Retentate 1Ultra Filtration 10 kD time=2h 36 minP=30 psi Total Protein=493.2 ug/ml

Filtrate 1Total Protein=19.82 ug/ml

Retentate 2Ultra Filtration 1 kD time=4h 14 minP=30 psi Total Protein=6.12 ug/ml

Filtrate 2Total Protein= 5.92 ug/ml

Ultrafiltration Flowchart

Reverse Phase HPLC

• Detects purity of Protein from injected sample

• Concentration can be found by integrating the peak area

• Protein X typically elutes around 15 minutes

The HPLC chromatogram

displays three elutions. The red line indicates a pure protein sample, the blue line represents pure protein from the cation exchange, and the black line portrays a sample of extract with spiked protein.

mV

-4.00

-2.00

0.00

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

HPLC Chromatogram

mV vs. Minutes

Final Discussion: The final results of this project have not been

determined, however, many progressive steps have been made in obtaining the protein from corn germ. Various extraction conditions (pH and salt conditions), filtration steps, and analysis of a quantification method (ELISA tests) are currently being researched to obtain a purified protein. The final steps of the protein sequence will be based on a combination of factors such as purity and yield which will be tested by reverse phase HPLC and the ELISA test.

Diagram of complete Antibody setup for ELISA testing

Acknowledgments:

An extended gratification and thank you is made to the multitude of people who not only made this project possible but successful:

• Dr. Charles Glatz• PWSE• Yandi Dharmadi, Zhengrong Gu, Maureen Griffin, Todd Menkhaus, and Jim Kupferschmit