1. Molecular Cloning of PylB, an Archaeal Lysine Isomerase
Katherine E Brandenstein, Williard J Werner, and Susan C Wang
School of Molecular Biosciences, Washington State University, Pullman, WA 99164
Introduction
The radical S-adenosyl-L-methionine (SAM) enzymatic
superfamily is believed to catalyze the largest variety of enzymatic
reactions as well as being the fastest growing superfamily under
investigation. This superfamily currently contains over
100,000 members, yet less than 50, only .01%, of the enzymes
have been studied in mechanistic detail.
Pyrrolysine (Pyl) is the twenty-second proteogenic amino acid and
is naturally found in methanogenic prokaryotes. Pyl derivatives
have been used in the medical industry for imaging, as probes for
ligation chemistry, and investigating enzyme mechanisms. The
biosynthetic pathway for Pyl includes the proposed radical SAM
enzyme PylB, which is thought to catalyze the isomerization of L-
lysine to form 3-methyl-D-ornithine using a radical SAM
mechanism. Understanding the PylB reaction may lead to altering
this catalyst so it can isomerize other amino acids for commercial
purposes.
Figure1. Proposed isomerization catalyzed by PylB.
The objective of my research was to create a synthetic DNA
construct to overexpress PylB in Escherichia coli so that it could be
mechanistically analyzed. Improving our understanding of the
catalytic process used by PylB will also improve our understanding
of other biosynthetic pathways. Eventually PylB may be used
together with additional enzymes from different pathways to create
new therapeutic compounds.
Figure 2 . Proposed radical SAM-dependent PylB isomerization mechanism.
Methods and Results
During my research experience I learned and used a variety of
molecular biology techniques.
After finding the construct of interest, we transformed E. coli
specially engineered for protein overexpression. We grew the cells
to check for protein overexpression using sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE). After collecting
~1 g of cells, they were moved into an anaerobic chamber for lysis
and purification. In the anaerobic chamber, I utilized an
immobilized metal affinity (IMAC) chromatography column to
attempt to isolate and purify the PylB protein. I ran additional SDS-
PAGE to confirm the production of PylB by my construct.
Future Possibilities
My cloning of the PylB overexpression construct will allow the
laboratory to generate large amounts of PylB, providing the chance
to study the natural PylB reaction as well as potential reactions
using different substrates. Future work will demonstrate the
catalytic process used by PylB, which can later lead to the use of
this protein to transform a variety of amino acids. Studies of PylB
can also improve the overall understanding of radical SAM enzyme
chemistry.
Acknowledgments
I was very fortunate to get a chance to learn a multitude of
laboratory techniques as well as a deeper overall understanding of
biology. This experience gave me confidence in my laboratory work
that I am able to bring to other labs during internships and future
schooling. Also, as I apply for graduate school this lab experience
has inspired me to explore the possibility of molecular engineering
programs. This work was sponsored by the Boeing Cyber Grant, The
Office of Multicultural Student Services, the Voiland College of
Chemical and Bioengineering, and the National Science Foundation
(CHE-953721).
References
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complete biosynthesis of the genetically encoded amino acid pyrrolysine from
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8. ExpressoTM T7 SUMO Cloning and Expression System. 2905 Parmenter St,
Middleton, WI 53562 USA.
1.Initially, to build
the DNA construct, I
planned to use a
DNA plasmid vector
denoted pET-24a(+)
to carry the PylB
gene.
2. To amplify my
desired DNA
segment, the PylB
gene from
Methanosarcinae
acetivorans, I used a
thermal cycler, an
instrument that runs
the polymerase chain
reaction (PCR),
which is the most common method for DNA amplification.
3. I restriction digested both the PylB gene insert and the
vector using the enzymes NdeI and NotI during the digest.
4. I then used DNA gel electrophoresis to purify the vector
and insert before adding DNA ligase to join the DNA
segments.
5. After ligation, I used electroporation, a method for opening
pores in bacteria through electric shock, to insert the DNA
construct into E.coli cells, and then grew the cells to find the
transformed construct.
Figure 3 The Expresso cloning system process. (8)
PylB PylB
PylB
PylB