Alexander E. Chu and Faculty Mentor: Eric N. Jellen, Department of Plant and Wildlife Sciences

Introduction
The central dogma of molecular biology teaches that the information of life is stored in
DNA, a linear chain of nucleotide molecules. The information in DNA nucleotides is then
transcribed into RNA, which encodes the amino acids needed to make proteins. These
chemically active proteins are responsible for nearly all the processes of life in the cell.
Thus, a single gene, consisting of relatively inert DNA, can give rise to a protein with a
highly specialized function, and the collection of genes contained in a genome can give
rise to the diversity of biomolecular function needed to sustain a living cell.
In many crop species, the CslF6 mixed-linkage glucan synthase is the protein most
responsible for the biosynthesis of mixed-linkage (1,3;1,4)-b-D-glucan (MLG). Naturally,
this compound is deposited in the cell wall and plays a role in determining plant
structure. However, when it is consumed, it becomes a nutrient critical for human
health, central in regulating blood glucose and cholesterol levels, mediating nutrient
uptake in the intestines, and even reducing the risk of colon cancer. Because oat is
unique amongst cereal crops in that it is usually consumed whole with the bran (unlike
other refined flours), it is particularly relevant in MLG research. Our lab’s expertise in oat
genetics further ensured that this project would best be carried out in oat (Avena sativa).
In this project, we sought to measure the reaction kinetics of the oat homolog of this
enzyme and identify key nucleotide changes that affect catalytic activity.
This project addressed questions with broad implications from both biochemical and
genetic perspectives. Most fundamentally, this represents the first attempt to generate
purified CslF6 protein, opening the door to a multitude of other potential studies,
including protein-protein interactions and crystal structure determination.

Methodology
This project was carried out in three main phases: 1) sequencing the homeoalleles of
CslF6 from A. sativa, 2) cloning these sequences into vectors for heterologous
overexpression in E. coli, and 3) kinetics assays on these sequences and the
interchanged sequences to determine enzyme efficiency and influential amino acid
residues.
We first sequenced the three homeoalleles of CslF6 from A. sativa and cloned the
soluble cytoplasmic domains of two of their respective gene products from the A and C
subgenomes into pET expression vectors.
Next, we attempted to express these sequences in two strains of E. coli as well as an E.
coli-derived cell-free expression system. Cell-free trials in the presence of proteinfolding
chaperones yielded a small amount of soluble, but inactive, enzyme. While this
was encouraging, it was insufficient for our downstream experimental needs, so we
continued to search for ways to express CslF6 in vivo. Further research led us to the
SIMPLEx expression system pioneered by the DeLisa lab at Cornell University.
SIMPLEx expression in SHuffle strains of E. coli yielded soluble protein, which we are
currently seeking to purify using an antibody against the polyhistidine tag attached to
the protein.

Results
Our work in genetically characterizing the homeoalleles of CslF6 from A. sativa and
their differential expression profiles is currently being prepared for publication in a peerreviewed
journal. Expression of CslF6 in SHuffle cells using SIMPLEx represents one of
the first experimental successes of the SIMPLEx system outside of the original paper.

Discussion
Our experimental work and research lays a foundation for imminent progress in
biochemically characterizing the oat CslF6 enzyme. Furthermore, because of its
complex, transmembrane nature, it is extremely difficult to express and therefore
characterize. To our knowledge, there exists no crystal structure of a plant cellulose
synthase. Thus, our breakthroughs are promising for future biochemical and structural
studies on not only CslF6 specifically, but the CES and CSL families of integral
membrane proteins.

Conclusion
Moving forward, we hope to elucidate the biochemical properties of the CslF6 enzyme.
Upon obtaining soluble, active recombinantly expressed CslF6 enzyme, we will conduct
calorimetric and spectrophotometric assays for biochemical characterization. Data from
these kinetics assays using UDP-glucose as substrate will be used to calculate km and
kcat values and solve the Michaelis-Menten equations for CslF6. Completing these
assays for both the A and C homeoalleles of CslF6 will clarify the effects of polypoid
variation on catalytic efficiency.
Once reaction kinetics and optimal conditions for both the A and C subgenome alleles
of CslF6 have been calculated, we will conduct site-directed mutagenesis experiments,
sequentially interconverting each non-synonymous single nucleotide polymorphism
(SNP) between the homeoalleles and assessing the change in enzyme kinetics and
catalytic efficiency for each change. Doing so will allow us to pinpoint a SNP or set of
SNPs that is responsible for differences in catalytic activity, demonstrating that genome
variation in polyploids can affect not only gene product expression and function, but also
chemical kinetics.