A genetically programmable protein module as intracellularly deliverable

QD-based FRET probes for viral protease detection

Nikola FinneranNikola Finneran

Divya Sivaraman, Payal Biswas, and Wilfred ChenDivya Sivaraman, Payal Biswas, and Wilfred ChenDepartment of Chemical and Environmental Engineering, Department of Chemical and Environmental Engineering,

University of California, Riverside, CA, 92521University of California, Riverside, CA, 92521

August 20, 2009August 20, 2009

Viral Proteases

Enzymes that catalyze the hydrolysis of peptide bonds

Very specific and highly expressed early on during infection

Cleave polyproteins into functional enzymes necessary for infection and disease progression

FRETFluorescence Resonance Energy Transfer

Energy transfer from excited donor to acceptor molecule

Spectral overlap of donor emission and acceptor absorption

Nature Reviews Molecular Cell Biology 4; 579-586

Fluorescent Protein-Based FRETFluorophores bound by specific linker protein

Disadvantages of using organic fluorophores and fluorescent proteins include:

narrow excitation bands

broad emission bands

low resistance to photo degradation

Hwang et al., AEM. 72(5): 3710–3715 (2006)

Quantum Dot-Based FRETAdvantages of using a quantum dot donor:

broad excitation bands narrow emission bands higher resistance to photo bleaching

Inability for intracellular delivery of the conjugated protein into cells

Nature Materials 5, 581 - 589 (2006)

QD-Based Engineered Protein Molecule

Modular protein design

CYS

Elastin-Like Protein (ELP)Repeating sequence {(VPGVG)2 (VPGKG) (VPGVG)2}20

Reversible temperature dependent precipitation

ELP

T>Tt T<Tt

ELP

T>Tt T<Tt

TAT Peptide

Allows QD-protein to penetrate mammalian cell walls

HIV-1 TAT peptide

Cluster of basic amino acids made up of 6 arginine and 2 lysine residues within a linear sequence of 9 amino acids (YGRKKRRQRRR)

Little to no cell toxicity

Protein ExpressionELP precipitation and centrifuging

48kD

CYS

Pure unconjugated protein molecule

QD and Alexa Dye ConjugatoinAlexa 568 maleimide dye conjugation with protein module

2 hour incubation of protein with Alexa 568 maleimide followed by thermal ELP purification

QD-Alexa Protein FRET Pair

QD : Alexa

Conjugated Protein Functionality

Conclusions and Future WorkModular peptide design – detect wide range of proteases

Low toxicity for in-vivo protease monitoring

Rapid protease detection

High throughput protease inhibitor screening

QDTAT

His6

CYS

cleavage site for PV2Apro

ELP QD

QD emission

after cleavage

AcknowledgementsNational Science Foundation (NSF)

Dr. Victor Rogers, Denise Sanders, and Jun Wang of the BRITE REU Program

Ph.D. candidates Divya Sivaraman, Payal Biswas, Divya Sivaraman, Payal Biswas, and Shen-long Tsaiand Shen-long Tsai

Professor Wilfred ChenProfessor Wilfred Chen

ReferencesHwang, Yu-Chen, Chen, Wilfred, Yates, Marylynn V. Use of Fluorescence

Resonance Energy Transfer for Rapid Detection of Enteroviral Infection In Vivo Appl. Environ. Microbiol. 2006 72: 3710-3715

Igor L. Medintz et al., Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates Nature Materials 5, 581 - 589 (2006)

Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto & Tullio Pozzan. Looking forward to seeing calcium Nature Reviews Molecular Cell Biology 4, 579-586 (July 2003)

Mahmoud Reza Banki, Liang Feng & David W Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags NATURE METHODS | VOL.2 NO.9 | SEPTEMBER 2005 | 659

Questions?

How it Works

Viral Protease

Virus

Cell-Wall