Significance/Background

Protein structure and function correlate together. Changing the structure of a protein will
almost always directly alter the function of
that protein. Learning about the structure of
a protein helps to also identify the function
of and protein a how changes in the
structure also change the protein’s function.
The smallest unit of protein is the monomer
amino acid. An amino acid is defined as a
simple organic compound containing a
carboxyl, amino group, and hydrogen all
connected to a central carbon with one
varying side group (Berg, 2002). These
monomers can be linked together to create
polymers, or protein.

There are four levels of structure

that are associated with proteins (Figure 1).
The first is the primary sequence which is
the simplest level of structure. This level
contains the sequence of amino acids linked
by peptide bonds into a polypeptide chain
and guides all other levels of protein
structure. The secondary level contains
alpha helices and beta sheets formed from
Figure 1: Four levels of protein structure
hydrogen bonding between the backbone of (Guru, 2017).
the polypeptide chain. The tertiary or third
level of structure is the initial level which
takes into account the varying side groups,
or R groups, of the amino acids. Disulfide bonds and non-covalent bonding between the R
groups of the amino acids creates the tertiary structure of the protein. The segments of protein
which fold independently and have their own functions in the tertiary level are called domains.
The last level of protein structure is quaternary structure. This level is made up of subunits. A
subunit is a single polypeptide chain which must join with other polypeptide chains in order to
produce a functional protein (Protein Structure, n.d.). An example of a protein with this level of
structure is the protein hemoglobin which has four subunits joined together to form functional
hemoglobin.

Although the basic facts of protein structure are known, it is still not clear for every
protein how changing the structure alters its function. Research related to protein structure and
function helps us to better grasp how structure affects the function of a protein. Any small
change in a single amino acid of the polypeptide chain sequence can have a large impact on the
function of that protein. Additionally, it has been found that there are various types of proteins
with various functions. Proteins can include things like antibodies, contractile proteins, transport
proteins, enzymes, and many more (Eight Types, n.d.). Furthermore, each of these types of
proteins have many subtypes. An enzyme, for example, has six types: oxidoreductases,
transferases, hydrolases, lyases, isomerases, and ligases (Martinez et al, 2015).

A type of hydrolase enzyme important in bioengineering is a nuclease, also referred to as

a restriction enzyme. Similar to how amino acids make up protein, nucleotides are linked
together by phosphodiester bonds
to create deoxyribonucleic acid
(DNA). The protein nuclease
catalyzes the hydrolysis of
nucleic acids (Nuclease, n.d.). In
simpler terms, the purpose of a
nuclease is to break the
phosphodiester bonds between
the nucleotides, Figure 2,
Figure 2: The breaking of a phosphodiester bond by a nuclease
creating a double-stranded break (Sequence).
in the DNA. Some nucleases are
known to be very specific as to where in a DNA sequence they will make a cut. These restriction
enzymes are found and harvested from bacteria which use them as a defense mechanism to
destroy viruses (Restriction Enzymes, n.d.).
Humans have similar digestive enzymes to
that of bacteria, but those enzymes have no
use in bioengineering as they cut DNA
randomly.

There are two major forms of

nucleases, exonucleases and endonucleases.
Exonucleases have the ability to remove
nucleotides one by one from DNA.
Endonucleases, on the other hand, cleave,
or cut, the phosphodiester bonds within the
DNA (Nucleases, n.d.). For the purpose of Figure 3: The sticky end residues left after DNA
cleavage using XHO1 and NDE1 restriction enzymes.
this research, endonucleases will be the
As seen, the cut sites are very specific for both
main focus. Similar to all enzymes, (XHO1) (NDE1).
endonucleases work by shape matching to
make cuts in DNA. This works when a specific part of the endonuclease, the recognition site,
recognizes a part of the DNA, the restriction site, where the shape matches that of the recognition
site on the endonuclease. The endonuclease then wraps around the DNA causing the break in
both strands of the helical DNA. These cuts are always in a predictable pattern as when an
endonuclease makes these cuts, it will leave overhangs in the DNA sequence called sticky ends,
as shown in Figure 3. These sticky ends are particularly useful in bioengineering as they allow
for a different DNA fragment to be inserted in the right direction (Restriction Enzymes, n.d.).

A common use in bioengineering for these endonucleases is to cut a whole section or

fragment of DNA out of what is called a plasmid. This is a small structure of circular strands of
DNA present in a bacterial cell’s cytoplasm which can replicate independently of the
chromosomes and have a common use in laboratory manipulation of genes. A new insert of
 DNA can then be inserted
into the missing section of
the plasmid to create
recombinant DNA, or
DNA that has been
artificially created by
combining constituents
from different organisms.
A common plasmid, or
vector, used in
bioengineering is pET-21a.
This plasmid is 5443 base
pairs long and contains the
gene encoding ampicillin
resistance (Figure 4). The
ampicillin resistance gene
is important for the
Figure 4: pET-21a plasmid showing the restriction enzyme cut sites bioengineering portion of
along with the ampicillin resistance gene (Novagen).
the plasmid as it allows for
the detection of the uptake of the plasmid into the bacteria.

Research using bioengineering tools to create recombinant DNA has produced a large
number of successful findings regarding the structure and function of proteins. Recombinant
protein research begins by understanding how altering a specific amino acid sequence alters the
folding of the protein and furthermore alters the structure and function for the protein. The
folding process is very complex and hard to understand without this slow process of altering
small sequences of the protein to see the effect on structure and function. The changing of DNA
sequences causes changes in protein sequences which might in turn change the function of the
protein that the DNA codes for. Synthetic protein design studies allow for further knowledge of
the fundamentals of proteins (Nollert, 2006). This research has been of special interest in recent
years, especially surrounding the idea of producing a small synthetically made endonuclease.
Synthetic restriction enzymes could be useful tools for diagnoses of diseases or as a potential as
drugs in pharmaceutical uses (Lim & Franklin, 2006). Different versions of this protein have
been successfully produced, but not in a cost-effective or easily-produced way. The process of
engineering a folded protein with a specific structure has been a significant difficulty in past and
current research. Creating proteins which have correct function and folding while introducing
metal cofactors for cleaving DNA remains an important milestone in synthetic protein design,
and even more specifically for synthetic enzymes.

The synthetic endonuclease in this study is referred to as a chimera, a hybrid protein

containing polypeptides with different functions by the fusion of two different parts of a protein
into one. This research pertains to a chimeric protein containing both the ability to bind to DNA
and cleave DNA sequences at a point of choice. This is useful as enzymes in nature that cleave
DNA are very site specific. The DNA binding portion is called the homeodomain which is
known as a helix-turn-helix motif, meanwhile, the metal binding portion is known as the EF-
hand motif. Neither the HTH nor the EF-hand motifs contain an enzymatic function of cleaving
DNA, but when fused together, this new function is produced. As previously stated,
endonucleases are very site specific, so creating one in a lab setting which can cleave DNA at a
chosen DNA sequence has been an interest in current research.

A domain has been

defined as a portion of the
polypeptide chain which folds
independently with a different
function. A motif, on the
other hand, is defined as a
supersecondary structure
which is a combination of
secondary structure elements,
like a mixture of alpha-helices
or beta-structures, by a loop.
The HTH motif, figure 5, is Figure 5: The helix-turn-helix of the homeodomain pairing with
two alpha helices joined the major groove of DNA (Lim & Franklin, 2006).

together by a short turn or

loop. This structure has the job of binding to DNA at the major groove. It is easy to tell apart the
major and minor grooves of DNA, as the major groove can be seen larger than the minor groove
by looking at the helix at a side angle.

The EF-hand motif is the most commonly-found motif in proteins that binds calcium,
shown in Figure 6. This motif is also composed of a helix-turn-helix structure similar to the
DNA binding homeodomain.
Proteins that bind calcium ions and
are involved in regulating activities
in cells are known as a calmodulin
proteins. The binding of a lanthanide
metal to the EF-hand motif has been
shown to hydrolytically cleave DNA
sequences, which is a function not
usually seen in calmodulin (Mallena
& Franklin, 2002). Phenomenally,
the similarity of the helices between
the HTH of the homeodomain and
the EF-hand motif has allowed for
these two different motifs to be
joined together by covalent bonding, Figure 6: Visualization of the EF-hand motif bound to a
while maintaining the function of the Calcium ion (Conley, 2015).
parent proteins (Mallena & Franklin,
2002).

Research by Welch et al. successfully created this chimera containing a calcium binding
loop (EF-hand motif) incorporated into the HTH motif of the homeodomain. This was produced
by splicing the calcium binding EF-hand motif of calmodulin into the HTH motif of the
homeodomain (Welch et al, 2001). The HTH homeodomain was used for a multitude of reasons,
including its small size, structure, and lack of cofactors, which is a chemical compound required
for enzyme activity to occur (Lim & Franklin, 2006). Due to these specific reasons, the helix end
of the EF-hand loop was able to be superimposed on the helix II and III of the homeodomain
 (Lim & Franklin,
2006). This
substitution allowed
for the turn portion of
the HTH to be
replaced with the
calcium-binding EF-
hand loop, fitting the
two pieces together
like a puzzle, Figure 7.
Figure 7: Computer design of the metallohomeodomain showing the The newly placed
chimera formed between the DNA binding homeodomain in green and
calcium-binding loop
the calcium-binding loop in orange (Welch et al, 2001).
had the ability to bind
lanthanides, allowing for hydrolytic activity due to the lanthanide metal’s ability to cleave
phosphodiester bonds (Welch et al, 2001). Lanthanide binds isostructurally in the calcium sites
on the protein, providing a way to deliver a hydrolytic metal to the DNA substrate due to the turn
substitution (Lim & Franklin, 2006). Lanthanide metals are naturally occurring metallic elements
labeled as atomic numbers 57 to 71 on the periodic table. In Franklin’s research, she helped
produce two generations of chimera proteins which had enormous impacts on the study of
protein structure and function along with the production of synthetic enzymes (Lim & Franklin,
2006).

Previous Research

The first prototype of chimeric peptides only contained the HTH of the homeodomain,
excluding the N-terminal arm and helix I to simplify the design (Welch et al, 2001). Then, the
EF-hand was successfully united with the HTH motif. This structure was confirmed using
nuclear magnetic resonance (NMR) to ensure the structural integrity was preserved (Welch et al,
2001). The first prototype had four different variations in structures named P1-P4. Out of the
four peptides created, only two, P3 and P4, were used for analysis. Proteins P3 and P4 were both
33-34 residue peptides long containing two helices, alpha 2 and alpha 3, however P4 differed by
not having the last turn of alpha 2 and no beta-turn. Both proteins were tested for their ability to
bind metal and hydrolytically cleave phosphodiester bonds in DNA. Upon these results, it was
found that P4 bound to Eu(III), a lanthanide, with higher affinity than P3 (Welch et al, 2001).
Eu(III), or Europium, is used as a luminescence probe to investigate the calcium-binding site
(Lim, 2006). It was found that P4 does not dimerize like P3, enabling for a higher binding
affinity due to the lack of dimerization. Gel shift assays of both P3 and P4 allowed for the
analysis of nuclease activity. A gel shift assay has the ability of showing DNA binding and
cleavage from the protein as the DNA band will shift on the gel when a protein is bound to it and
when the DNA is cleaved (Gel
Shift, n.d.). DNA cleavage was
found to be lesser in P3 than in
P4, further justifying the dimer
formation in P3 (Welch et al,
2001). Luckily, P4, was found to
possess both the ability to bind
calcium and cleave DNA
indicating that this protein
building approach can
successfully be used to create
proteins with novel functions.
The engineering of these proteins,
however, was completed using a
significantly expensive solid state Figure 8: The P4 structure of the EF-hand of calmodulin (top)
synthesis machine rather than and the HTH homeodomain (bottom) which were constructed
together to form the P4 protein (Sirish & Franklin, 2002).
using cheaper recombinant DNA
and protein expression in bacteria (Welch et al, 2001).

The first P4 protein was generated in January of 2001 and another version of P4, shown
in Figure 8, was produced in November of 2001. The aim of the second P4 study was to
determine the effect of a flexible peptide on nuclease activity and test the affinity of P4 to bind
Ce(IV) rather than Eu(III) (Mallena & Franklin, 2002). Similar to Eu(III), Ce, or Cerium, is a
lanthanide metal found naturally on earth. One main aim to improve the P4 design was to
decrease the flexibility of the protein. It was suggested that a rigid structure may be a preferable
design of selectivity for the binding of metals. The origin P4 was found to have improper
structure upon binding to lanthanide metals, so a major goal for the new P4 was to create a less
flexible chimeric protein. In this version of P4, the peptide was shown to bind Eu(III) with lesser
affinity than the first one. It did, however, have a higher binding affinity for Ce(IV), making it
the first peptide to show catalytic activity in a Ce(IV)-EF-hand motif. Researchers hypothesized
this better binding affinity for Ce(IV) was due to charge difference in the two ions. To ensure the
cleavage of DNA was not occurring spontaneously, cleavage was tested using P4 not bound to a
lanthanide metal. Results concluded that hydrolytic activity was significantly increased in the
presence of P4 bound to
either a Eu(III) or Ce(IV) ion
(Mallena & Franklin, 2002).

After the production

of the P4 protein, Lim and
Franklin created a second
generation of chimera
proteins in 2006 (Lim &
Franklin, 2006). These were
named C1-C4 and used the
same EF-hand substitution for
the turn portion of the HTH
motif of the homeodomain
(Figure 9). This generation of
chimeric proteins were Figure 9: The four models of the second generation of
created in a more cost- metallohomeodomains (C1-C4) (Lim & Franklin, 2006).

effective way using

recombinant DNA in a laboratory setting. Rather than the small 33-34 amino acid long peptide
used to create P4 previously, the second generation contained an extra 30 residues on the N-
terminus of the homeodomain (Lim & Franklin, 2006). Upon purification, C1 and C3 denatured,
which was, in this case, most commonly caused by misfolding. C2 and C4, on the other hand,
were purified and used for analysis. It was discovered that C2 had a secondary structure that was
very similar to the parental P3 and P4 proteins previously produced. Also, C2 had improved
lanthanide-dependent folding and stability compared to the smaller peptide design. Despite this
improvement, C2 showed little to no hydrolytic activity towards DNA. According to Lim and
Franklin, a possible explanation for the lack of hydrolytic activity was due to the twist of a
helical bundle being inverted, distorting the metal binding site causing the lanthanide-center to
be inaccessible to the DNA substrate for cleavage (Lim & Franklin, 2006).

Research conducted by Loras Even at Loras College in 2008 showed results suggesting
that the P4 protein had been recreated using recombinant DNA synthesis. Although tests showed
that the construction had been successful, Loras was left with many unanswered questions about
the structure of the protein after leaving Loras College. Unanswered questions included needing
to test the insert by cutting it out of the plasmid using XHO1 and NDE1 restriction enzymes, the
enzymes originally used to cut the plasmid for the insert to be ligated into. This would ensure the
correct insert was placed into the plasmid. Also, isolation of the DNA from DE3 bacterial cells
would need to be performed in order to obtain the DNA so it could be sent to a sequencer (Loras,
2008). Taylor Keeney, another student at Loras College, continued this research seven years later
in 2015. Taylor also had speculation that he had produced the P4 plasmid with the correct DNA
sequence to code for the P4 protein, however, these results were shown to be false using gel
electrophoresis and UV spectrometry. Although growth had occurred after transformation
signifying that the plasmid was taken into the bacteria, the gel electrophoresis and enzyme test-
digests after isolation confirmed this isolated DNA was not the engineered plasmid. This false
positive most likely resulted from contamination (Keeney, 2015).

Research Goals

In this research, I hope to combine and fix the issues between the past chimeric protein
generations. The first was too expensive, but did properly fold, bind lanthanides, and
hydrolytically cleave DNA. The second (C proteins) lacked the hydrolytic activity, but was
created in a less expensive procedure. Similarly to past students, but hopefully with more
success, I will be recreating the P4 protein using recombinant DNA synthesis, rather than solid-
state synthesis, to produce a protein which binds lanthanides, folds properly, and shows
hydrolytic activity while using a cost-attainable procedure. The recombinant procedure will
include using specific restriction enzymes to cleave pET-21a DNA. The remaining vector will
then have sticky ends which will be used to insert the DNA encoding the chimeric protein. Once
fully produced and in DE3 bacteria, the sequence can be ensured as correct by sending the
plasmid in to be sequenced after being isolated from the bacterial cells it resides in. Then, a gel-
shift assay can be ran to ensure this protein contains the DNA binding and catalytic cleaving
capability. This would provide a cost-effective way of successfully producing a functional
synthetic enzyme which has possible future use for pharmaceutical applications. Furthermore,
success in this research will further help us understand protein structure and function by having
the option to further study how changes in amino acid structure changes protein function.

Methods

The methods used during this researching process were those involved in recombinant
DNA production. These involved methods involved purifying, cleaving, and transforming DNA
using DH5 alpha bacteria, a type of Escherichia coli (E. coli). The ending product of desire was a
metallopeptide with the ability to cleave DNA sequences of self-selection.

Preparing 500 mL Luria Broth

In a 1 L Erlenmeyer flask, 5 grams of NaCl, 5 grams tryptone, and 3.5 grams of yeast
extract were added. Then, water was added to the 500 mL mark on the flask. The flask was
stirred until all solids were dissolved. The flask was then topped with cotton wrapped cheese
cloth which was covered with tinfoil. The flask was placed in the autoclave on slow for 30
minutes for sterilization.

Preparing 500 mL Luria Broth Agar Transformation Plates (With and Without Ampicillin)

The same procedure was completed from Preparing 500 mL Luria Broth with the
addition of 7.5 grams of agar. After sterilization, the solution was allowed to cool enough to be
able to touch. Then, the solution was poured into plates and became transformation plates which
contained no antibiotic. Then, for ampicillin plates, 1 microliter of ampicillin was added per 1
mL of LB agar left in the flask. This solution containing the antibiotic was poured into plates and
became ampicillin containing transformation plates.

Preparing an Electrophoresis Gel

In a flask, 0.4 grams of agarose was added to 50 mL of 1X TAE buffer prepared and kept
in the DNA lab by the lab technician. The mixture was heated by 10 second increments in the
microwave until a clear solution remained. Once cooled to a touchable temperature, 50
microliters of ethidium bromide (1 microliter ethidium bromide per 1 mL) was added to the flask
and swirled. This was then poured into a mold until solidified.

Cleaving pET-21a DNA Using XHO1 and NDE1

In a sterile centrifuge tube, 3.33 microliters of 300 ng/microliter pET-21a DNA, purified
by a past student was added. Then 2 microliters of cutsmart buffer, 0.5 microliters of XHO1
enzyme, 0.5 microliters NDE1 enzyme, and 13.7 microliters of sterile distilled water was added
so the solution totaled 20 microliters. The tube was placed in a circulating water bath at 37˚ C for
one hour. This was then run on an agarose gel to analyze the enzyme activity. On the gel,
controls were used, which included loading one lane on the gel with uncut pET-21a DNA and
then two lanes loaded with single cut pET-21a DNA, one with only XHO1 enzyme and the other
with only NDE1 enzyme.

DNA Purification from an Agarose Gel

The double digested DNA, cut with both XHO1 and NDE1, was extracted from the gel.
The gel was placed under ultraviolet light to be analyzed and the band containing the double
digested DNA was cut out using a clean razor. This was placed into a sterile centrifuge tube.
Then, a gel extraction purification was completed using the Qiagen gel extraction kit. The
purified DNA concentration was tested using a Take 3 test on the DNA lab computer.

Annealing Oligonucleotides

The oligonucleotides (oligos) were purchased previously and are short pieces of DNA
which are complementary to one another and contain the sequencing for the metalloprotein
attempting to be produced. The oligos were annealed together by placing 1 microliter of each
oligo, the forward and backward, into a sterile centrifuge tube with 48 microliters of annealing
buffer. The annealing buffer was produced by adding 0.058 grams of NaCl with 0.109 grams of
Hepes into 8 mL of sterile distilled water. The pH was adjusted to 7.4 and the buffer was
sterilized by filtration. The centrifuge of oligos and buffer was placed at 90 degrees Celsius for 5
minutes and then cooled down slowly to room temperature by taking the heat block out of the
incubator and placing it onto the lab bench. Once cooled, the annealed oligos were placed in the
refrigerator.
Ligation of Annealed Oligos into Double Digested pET-21a DNA

5 microliters of the double digested DNA was placed into a centrifuge tube with 1
microliter of ligase, 2 microliters of ligase buffer, and 12 microliters of distilled water. The
centrifuge tube was kept at room temperature for 10 minutes. It was then transferred to a heat
block at 65 degrees Celsius for 10 minutes to heat kill the ligase enzyme. This ligated DNA was
then placed into the freezer to use in transformation.

Preparing Competent Cells

250 mL of transformation buffer was produced first by mixing 3.75 mL 1 M CaCl2, 27.5
mL 0.5 M MnCl2, 25 mL 2.5 M KCl, 5 mL 0.5 M Pipes at pH 7, and 188.8 mL of distilled water.
The CaCl2, MnCl2, KCl, and Pipes solutions were made prior by the use of calculations to get the
correct end concentration. The transformation buffer was sterilized through vacuum filtration and
then kept in the DNA lab refrigerator. In a 1 L flask, 250 mL of Luria broth was produced and
1.25 mL of 2 M MgCl2 was added along with a pre-culture of DH5 alpha E. coli cells. This
culture of cells was allowed to grow overnight. The rest of the procedure was completed on ice.
The cells were transferred to six cooled 50 mL conicals and chilled on ice for 10 minutes. The
six tubes were then centrifuged at 0 degrees Celsius for 10 minutes at 5000 rpms. The liquid was
decanted. Using 15 mL of transformation buffer, one conical of cells were re-suspended and then
dumped into another conical. This procedure was repeated twice more until three conicals of
cells were placed into one conical with 45 mL of transformation buffer. This procedure was
repeated for the other three conicals. The two conicals were centrifuged again for 10 minutes at
5000 rpms and 0˚ C. The liquid was decanted. The cells were re-suspended in 20 mL of
transformation buffer for each conical. 1.5 mL of DMSO, located in the DNA lab refrigerator,
was then added to each of the two conicals. From the two conicals, 500 microliter aliquots were
made into centrifuge tubes and placed into liquid nitrogen for quick freezing. After all the
aliquots were made, all the competent DH5 alpha cells were placed in the -80˚ C freezer.

Transformation of pET-21a DNA into DH5 alpha Cells

One 500 microliter aliquot of competent cells was placed on ice to thaw. Once thawed, a
100 microliter aliquot was immediately placed into a centrifuge tube that was cooled on ice. Into
the 100 microliters of cells, 10 microliters of the previously ligated DNA was added. This ligated
DNA was the purified double digested pET-21a DNA that was ligated with the annealed oligos.
The centrifuge tube was flicked 4 times to mix. The tube was left on ice for 20 minutes. While in
the ice, a circulating water bath was set to 42˚ C. After 20 minutes on ice the tube was placed
into the 42˚ C water bath for 45 seconds and then placed back on ice for 5 minutes. Then, 890
microliters of LB broth was added to the centrifuge tube to equal a total of 1000 microliters. This
tube was placed into a small flask that was placed into the shaking incubator at 37˚ C for 30-45
minutes. In addition to the flask with the transforming centrifuge tube, an ampicillin plate was
placed into the incubator at the same time to pre-warm the plate for transformation. After 30-45
minutes, the flask with the centrifuge tube along with the ampicillin plate were removed from the
incubator. The centrifuge tube was placed into a centrifuge for 4 minutes at 2000 RPMs. All but
100 microliters of the liquid was decanted from the tube. The cells were then resuspended in the
remaining liquid in the centrifuge tube. The complete solution in the centrifuge tube was put on
the pre-warmed ampicillin plate. The plate was taped shut, inverted, and placed in a 37˚ C
incubator for 12-16 hours.

Limitations

Some limitations that occurred during this research pertained to the ability to keep
sterilization during the duration of the project. The pipette tips being used throughout the
research were being used by many different students. Although the tips are sterilized prior to use,
it is likely that this equipment was left open, failing proper sterilization techniques. This lack of
sterilization caused false positive growths to occur on ampicillin containing transformation plates
leaving it to be believed that transformation had been successful when it actually hadn’t been.
The transformation was proven false by having the bacteria grown purified and sent to the
University of Iowa for sequencing to see if the sequence matched that of the oligos or pET-21a
DNA.

In addition to the tips lacking sterilization at all times, it is likely that the pipettes being
used were always calibrated properly. This could have caused deviations in the amount of
various solutions being used caused skewed results as precision and accuracy on volume delivery
is important to ensure proper results. Again, because of various classes using this piece of
equipment it wasn’t possible to have a set of pipettes calibrated only for this research’s use.
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