A HIGH-THROUGHPUT LINEAR EXPRESSION TEMPLATE SYSTEM

FOR ANALYZING PROTEIN ACTIVITY AND STABILITY

MARK LINDSAY, JEFFREY WU, BRADLEY C. BUNDYDept. of Chemical Engineering

Brigham Young University

Utah Conference of Undergraduate ResearchFebruary 28, 2014

What are proteins?

Basic building blocks of lifeNecessary for body functions

Metabolism Immune system DNA

Made up of amino acids

Bioengineering Proteins

Protein research has already improved Catalysts Drugs Vaccines

Protein production market valued at over $100 billion annually.

Unnatural Amino Acids (UAA’s)

20 universal amino acidsMore combinations = more biomedical,

pharmaceutical, and catalysis products

Problems with UAA incorporation

1- Difficulty of Incorporation2- High time cost3- Effects on protein structure

Simplifying UAA Incorporation: Cell free protein Synthesis

N3

Cu(I) N NN

DNA of Protein of Interest, Amber

Codon Replacement

UAA tRNA Synthetase

UAA

LyseLysis

Add

Linear Expression TemplatesImproving Time Efficiency

Polymerase Chain Reaction (PCR)`

(http://users.ugent.be/~avierstr/principles/pcr.html)

Linear Expression TemplatesImproving Time Efficiency

1 Week

1 Day

Protein Structure

Protein activity is highly dependent on structure

Changing Amino Acids can significantly affect structure

Example: Sickle Cell Anemia

Choosing With a Solved Structure

Active Site

Site far away from active site

Site near active site

Immobilized Enzyme Activity Data

0 10 20 30 40 50 600

5000

10000

15000

20000

25000Random Attachment (at primary amines)

Attachment Near Active Site (T21Amb)

Attachment Near Active Site (K35Amb)

Time (min)

Flu

ore

sc

en

ce

(rf

us

)

What To Do With Unknown Structure

• Actual Structure Bronze• Prediction Software Blue

Current Work: High-Throughput Testing

Create UAA mutations at dozens of selected sites

Simultaneously test effect of UAA incorporation on: Protein activity Protein stability

Acknowledgements

Research Advisor Dr. Brad Bundy

Graduate Students Jeff Wu

Undergraduates Chris Werner Chris Hutchings Steven Stanley Justin Crandall

Funding DARPA YFA NSF CAREER NASA NPB BYU

Bundy Lab Presentations at UCUR

Bennett A, Smith MT, Bundy BC. Foot-and-Mouth Disease Vaccine: Technical and Political Challenges to Vaccine-based Eradication. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.

Hunt J, Smith MT, Bundy BC. Cell-free Unnatural Amino Acid-Incorporation using Linear Expression Templates. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.

Hutchings C, Wu JC, Bundy BC. Immobilizing Biocatalysts onto Surfaces. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.

Werner C, Bundy BC. Investigating Linear DNA Expression Templates. Utah Conference on Undergraduate Education. Brigham Young University. Feb 2014.

Questions?

PCR Mutagenesis Techniques and RTS

Site Specific

Current methods of uAA incorporation are random sites or either beginning or end

We can add site specificResults indicate advantages of site

specific