(620bw) Using Sacrificial DNA to Improve LET-Based Cfps Protein Yields

Using Sacrificial DNA to
Improve LET-Based CFPS Protein Yields

Andrew Broadbent,
Bradley C. Bundy

Brigham Young
University, Provo, Utah, USA

AIChE 2015 Conference
Abstract

Proteins?polymers of
amino acids?are a major class of biomolecules whose myriad functions facilitate
many crucial biological processes. Accordingly, human control over these
biological processes depends upon the ability to study, produce, and modify
proteins. One innovative tool for accomplishing these aims is cell-free protein
synthesis (CFPS). This method, rather than using living cells to make protein,
simply extracts the cells' natural protein-making machinery and then uses it to
produce protein in vitro. Because living cells are no longer involved,
scientists can freely adapt the protein production environment in ways not
otherwise possible. However, improved versatility and yield of CFPS protein
production is still the subject of considerable research.

This work seeks to
improve CFPS protein yield by preventing the degradation of template DNA in
CFPS. Among the advantages of CFPS is the option of using linear expression
templates (LETs) in place of plasmids as the DNA template for protein
production. Because LETs can be produced more quickly than plasmids can, using
LETs greatly reduces the time required to obtain a DNA template for protein
production. This renders CFPS a better candidate for high-throughput testing of
proteins. However, LETs are more susceptible to enzyme degradation than
plasmids are, which means that LET-based CFPS protein yields are lower than
plasmid-based CFPS yields. This work explores the possibility of increasing the
protein yield of LET-based CFPS by addition of sacrificial DNA, DNA which is
not used as a protein-making template but which is degraded by the enzymes in
place of the LETs.