1. Researchers developed a Si-tag fusion protein containing Si-tag, RGD, and His-tag to enable delivery of biomolecules like RGD onto silica surfaces for tissue engineering applications. 2. They expressed the fusion protein in E. coli and tested various purification methods, finding that rapid purification using silica nanoparticles was more effective than silica gel or IMAC. 3. Functionality assays using immunostaining and binding to silica nanoparticles confirmed the fusion protein retained its ability to bind silica surfaces. Further optimization of purification conditions is still needed to improve protein yield.

1. Optimization of Purification and Functionality of Si-tag Fusion Protein for Tissue Engineering
Toby Yang, Megan Thor, Adam Hildebrandt, Eric Heimendinger
Faculty Advisor: Dr. Mary Ann Yang, PhD
Concordia University, St. Paul
Background
Neurodegeneration is the progressive loss of structure or function of neurons, which includes death of neurons. There are hundreds of various neurodegenerative diseases that remain a mystery to
science and medicine but the forefront of attention has been given to only a few such as Alzheimer disease (AD), Huntington's disease (HD), Parkinson disease (PD), and amyotrophic lateral sclerosis
(ALS). The central nervous system (CNS) has a low intrinsic capacity for spontaneous regeneration following injury or disease, but neural stem cells (NSCs) demonstrate pluripotent potential to
differentiate into various types of neuronal cell for treating brain injuries or neurodegenerative diseases (Chen et al., 2013) like PD. The success of NSC transplantation in animal models is difficult to
translate when attempted in clinical conditions since the competence of the transplanted stem cell is limited due to low cell survivability and integration rates (Thuret et al. 2006). In order to increase
stem cell survivability and integration rates it is important to utilize biomaterials that are nontoxic whilst coaxing the NSCs to proliferate properly. The field of tissue engineering combines three-
dimensional (3-D), bioactive, and biodegradable scaffolds, cells, and regulatory molecules to create ideal bio-mimic microenvironments for restoring and regenerating injured body tissues (Chen et al.,
2013; Feng et al., 2016). Silica nanofibers (SNFs) are inorganic-based biomaterials that have been shown to be capable of providing good three-dimensional support and guidance for the growth of
NSCs (Chen et al., 2013). The SNFs mimic the extracellular matrix (ECM) and act as the scaffold for growing NSCs but the ability to freely control and deliver different biologics onto that scaffold to
interact with the cells is incredibly important for the field of science and medicine.
Mammalian cells have a protein known as integrin on their membrane and many integrin’s are known to bind proteins that contain the three amino acid sequence of Arg-Gly-Asp (RGD). Ribosomal
protein L2 found in E. coli was shown to have a strong affinity for binding silica surfaces and was designated the name Si-tag (Taniguchi et al., 2006). Since Si-tag can be engineered as a fusion protein
to enable the delivery of other proteins onto silica surfaces, in this project we hypothesize that Si-tag can be utilized in a way to get cells to adhere onto glass surfaces. We propose this can be
accomplished by fusing Si-tag with RGD, while Si-tag binds the glass surface; RGD binds onto the mammalian cell and can be cultured.
In this study we utilized genetically engineered Rosetta DE3 pLysS E. coli transformed with the plasmid pET21 that codes for Si-tag*RGD*His-tag to produce the fusion protein. The purpose of the Si-
tag protein is to allow the entire fusion protein to sit on glass/silica surfaces, which would enable the delivery of any desired biologics attached to Si-tag, in this case it would be RGD. RGD as mentioned
earlier will interact with the integrin proteins on mammalian cells and allow the cells to settle on the glass surface. The purpose of His-tag was to allow us more mechanisms in which to detect and
confirm the presence of the fusion protein Si-tag*RGD*His-tag.
A B C
Figure 3. Immunocytochemistry (ICC) assay for the functionality of Si-tag fusion protein on glass coverslips.
Si-tag*RGD*His-tag fusion protein collected from Rosetta pLysS E. coli transformed with pET21, aliquot on glass coverslip and washed with
High Salt TBST and incubated with Anti-His antibody/2% BSA and Anti-Mouse Alexa Fluor 488 Goat antibody/2% BSA. Images were
magnified by 20x with exposure at 350ms. (A) Control group from Rosetta only containing non-IPTG induced protein. (B) 0.1 mM IPTG
induced protein, (C) 1 mM IPTG induced protein.
Functionality Assay of Si-tag Fusion Protein
Figure 2. SDS-PAGE and Western Blot of Si-tag fusion protein expression. Rosetta pLysS E. coli transformed with pET21 Sitag-RGD-Histag were induced with
IPTG, lysed, centrifuged and the samples collected were assessed by (A) SDS-PAGE and (B) Western Blot. Sample 1 (S1) were induced with 0.1mM IPTG and the
lysed mixture were spun at 5000rpm. Sample 2 (S2) were induced with 0.1mM IPTG and the lysed mixture were spun at 4000rpm. Sample 3 (S3) were induced with
1mM IPTG and the lysed mixture were spun at 4000rpm. NI: Non-induced sample, SUP: lysed supernatant after induction, Pellet: lysed pellet after induction.
S1:$NI$
S1:NI
Ladder
Ladder
S1:SUP
S1:SUP
S1:Pellet
S1:Pellet
S2:NI
S2:SUP
S2:$Pellet$
S2:NI
S2:SUP
S2:Pellet
S3:NI
S3:SUP
S3:Pellet
S3:NI
S3:SUP
S3:Pellet
A" B"
0.1mM IPTG
5000rpm
0.1mM IPTG
4000rpm
1mM IPTG
4000rpm
kDA$
~135$
~75$
~63$
~48$
~35$
~25$
~17$
~11$
0.1mM IPTG
5000rpm
0.1mM IPTG
4000rpm
1mM IPTG
4000rpm
Expression of Si-tag fusion protein
The Process
Ribosomal protein L2 is an intrinsic protein found in E. coli and has the ability to bind silica. The
commercialized name for this protein is Si-tag. Research shows that silica nanofibers are degradable in PBS,
which is an important characteristic for scaffolds used in tissue engineering. These two findings can be
combined to bring about a new and novel approach to tissue engineering utilizing Si-tag fusion protein to
deliver not only stem cells onto the scaffold but possibly a multitude of desired biologics onto the scaffold
surface. To utilize Si-tag as the delivery mechanism it must be purified, which is possible through high salt
elution but is not physiologically favorable hence the new and novel method utilizing a rapid purification
mechanism.
Figure 9. Silica-binding proteins
in E. coli. Lane 1, cleared
supernatant prepared from cell
lysates. Lane 2, proteins that
bound to silicon particles in the
presence of 0.5% Tween and 1
M NaCl.
Figure 10. SEM images of SNF (A and D), immersed in
10 mM PBS for different time periods at 37 ℃: (A) after 6
days. (D) after 11 days. Inset in D is the magnification of
the corresponding surface. Scale bar represents 1 µm in
A and 100 nm in D. Abbreviations: SEM, scanning
electron microscopy; PBS, phosphate-buffered saline;
SNF, silica nanofiber.
Figure 11. Dissociation conditions of Si-tag from silica.
Silica particles with the bound Si-tag were suspended and
incubated for 10 min in the following solutions at room
temperature: 50 mM Tris buffer (pH 8.0) containing 5 M
NaCl (lane 2), 2 M MgCl2 (lane 3), or 2 M CaCl2 (lane 4); 1
N HCl (lane 5); 1 N NaOH (lane 6). Silica particles were
collected by centrifugation, and then the Si-tag still bound to
the particles was analyzed by SDS–PAGE (12.5%). Lane 1
is a control without the above elution procedure. Lane M,
molecular mass markers. Proteins were stained with CBB
R-250.
Figure 12. Rapid purification of Car9-tagged proteins
with a disposable device. A: Schematic representation
of the device. B: SDS–PAGE analysis of clarified lysate
from cells producing GFPmut2-Car9 before loading (L)
and after lysine elution (E).
LSupFTW1W3SGCE
SG1hour
CE1hour
Figure 7. Western blot of rapid purification of Si-tag fusion protein. Si-
tag*RGD*His-tag fusion protein collected from Rosetta pLysS E. coli
transformed with pET21, purified using 3 grams of 60-200 silica mesh and
assessed by Western blot. L: Ladder, Sup: Supernatant, FT: Flow Through, W1:
Wash 1, W3: Wash 3, SG: Silica gel (3g), SG 1 hour: Silica gel incubated for 1
hour in buffer, CE 1 hour: Concentrated Eluent from 1 hour incubation, Buffer:
20mM Tris-HCL pH 7.5, Elution: Buffer + 2M L-Lysine
Ladder
SUP
FT
Wash1
Wash2
Eluent
Silicamesh
48
35
25
17
11
kDa
63
75
Figure 8. Si-tag fusion protein assessed by SDS-Page gel. Si-tag fusion protein induced
at 0.1mM IPTG, lysed and collected via. rapid purification using a commercialized kit then
assessed by SDS-PAGE. SUP= supernatant, FT= flow through.
Optimization of Rapid Purification
Figure 4. SDS PAGE of Silica Nanoparticle (SNP) Functionality Assay. Rosetta pLysS E. coli transformed with pET21 Sitag-RGD-Histag
were induced with IPTG, lysed, centrifuged and incubated with Silica Nanoparticles, and were assessed by SDS-PAGE. Control SUP: Non-
Induced supernatant after lysis, E1: Induced supernatant after lysis, E2: Induced supernatant after lysis
Ladder
Ladder
E1Lysate
E1FlowT
E1Wash1
E1Wash5
E2Lysate
E2FlowT
E2Wash1
E2Wash5
E2Pellet
E2Lysate
E2FlowT
E2Pellet
CLysate
CFlowT
CWash1
CWash5
CPellet
kDA
~135
~75
~63
~48
~35
~25
~17
~11
kDA
~135
~75
~63
~48
~35
~25
~17
~11
1mM IPTG SUP0.1mM IPTG SUP 1mM IPTG SUPControl SUP
1. Taniguchi, K., Nomura, K., Hata, Y., Nishimura, T., Asami, Y., & Kuroda, A. (2007). The Si-tag for
immobilizing proteins on a silica surface. Biotechnology and Bioengineering, 96(6), 1023–1029. https://
doi.org/10.1002/bit.21208
2. Ikeda, T., Ninomiya, K., Hirota, R., & Kuroda, A. (2010). Single-step affinity purification of recombinant
proteins using the silica-binding Si-tag as a fusion partner. Protein Expression and Purification, 71(1), 91–
95. https://doi.org/10.1016/j.pep.2009.12.009
3. Coyle, B. L., & Baneyx, F. (2014). A cleavable silica-binding affinity tag for rapid and inexpensive protein
purification. Biotechnology and bioengineering, 111(10), 2019-2026.
4. Feng, Z. V., Chen-Yang, Y., Chen, W. S., Keratithamkul, K., Stoick, M., Kapala, B., … Yang, M.-L. (2016).
Degradation of the electrospun silica nanofiber in a biological medium for primary hippocampal neuron
– effect of surface modification. International Journal of Nanomedicine, 729. https://doi.org/
10.2147/IJN.S93651
References
Objectives
M Lysate Flow W1 W2 W3 E1 E2 E3
Figure 5. Si-tag fusion protein purified via. Immobilized Metal Affinity
Chromatography (IMAC) and assessed by SDS-Page gel. Si-tag fusion
protein collected by induction, lysis and IMAC purification using a Nickel
column. The fusion protein has eluted, but there are still residual proteins
in the samples.
Immobilized Metal Affinity Chromatography (IMAC)
Purification
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Past Present Future
● To test the induction of the
fusion protein in E. coli using
isopropyl β-D-1
thiogalactopyranoside (IPTG),
a molecule that mimics
allolactose and induces the
transcription of the DNA
necessary to synthesize our
Si-tag fusion protein.
● To test the purification of
Sitag-RGD-Histag using Nickel
ion IMAC.
● To test the functionality of
Sitag-RGD-Histag and its
affinity to silica glass
coverslips using
immunocytochemistry (ICC).
● To test the Si-tag fusion
protein's ability to bind onto
silica nanoparticles (SNP).
● To test the purification of Si-
tag through SNP using High
Salt elution.
● Testing of primary neuron
growth on silica surfaces
coated with the Si-tag fusion
protein.
• Optimize purification of Si-tag
fusion protein
How?
Rapid Purification
Silica Gel amount to
sample amount ratio
Utilizing SNP instead of
Silica Gel
Test different Incubation
time with buffers
Amicon Filter-to
concentrate
Utilizing 2M L-Lysine as
elution buffer
Test the functionality of Si-tag
fusion protein by:
Immunocytochemistry (ICC)
Silica nanoparticle (SNPs)
Purification optimization
Utilize SNP instead of Silica
Gel.
Test High Salt + L-Lysine
combination as elution
buffer.
Test different incubation
times of lysate and SNP
and SNP + elution buffers.
Grow Neuronal Cells
Coat SNF with purified Si-tag
fusion protein.
Seed with cells (Test scaffold
with PC12 cells).
Detection tests of Si-tag
fusion protein after cell
adherence.
Figure 3 shows the functionality assay of the Si-
tag fusion protein utilizing the Anti-His antibody,
which detects the epitope of His-tag on the Si-tag/
RGD/His-tag fusion protein. AlexaFluor488
antibody detects the Fc portion of the Anti-His.
Using fluorescent microscopy, the fluorescent
signals are detected. Horse Radish Peroxide
(HRP) antibody can also be used to detect the
fusion protein via. western blot as seen in Figure 3
and 6. Figure 3 shows a green fluorescent smear
across the glass surface, which indicates the Si-
tag fusion protein is functional, however, this test
is not always consistent and so a secondary assay
utilizing silica nanoparticles (SNPs) was performed
as shown in Figure 4. Figure 4 shows that the Si-
tag fusion protein is functional indicated in the lane
containing the pellet. Figure 5 shows the success
of rapid purification of the Si-tag fusion protein,
however, most of the fusion protein is collected in
Wash 1 and Wash 3. Since the purification
apparatus utilized silica gel as the binding surface
for Si-tag we hypothesize that the gel has simply
too small of a surface area to allow all of the Si-tag
fusion protein to bind to and is the reason why
most of the Si-tag fusion protein is detected in
Wash 1 and 3. The buffer and elution system
utilized by Coyle et al. had great success in
purifying the fusion protein Car9-GFPmut2. The
method of purifying Car9-GFPmut2 utilizes Car9's
ability to bind silica, though not as strongly as Si-
tag, this method allowed for the successful
purification of this fusion protein. We hypothesized
that by utilizing their buffer and elution systems we
would be able to elute more of the Si-tag fusion
protein and collect it in pure fractions. Figure 6
shows that the Si-tag fusion protein was able to
bind to the silica mesh; however, it was not
successfully collected into the eluent after washing
and eluting utilizing Coyle et. al’s buffer and
elution systems. It may be that a stronger eluent is
needed to purify the Si-tag fusion protein off silica
surfaces.
Previous
Experiments
Results
His-tag
purification
using Nickel
Column
Si-tag
purification
using SNPs
eluted with
MgCl2
Si-tag
purification
using Silica
Mesh eluted
with 2M L-
Lysine
His-tag Protein
did not bind
effectively
Desalting of
2M MgCl2 was
laborious and
difficult
Still optimizing
M NI 30minS 30minP 60minS 60minP 90minS 90minP
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Figure 1. SDS-PAGE Results from Testing Different IPTG Induction Times. A SDS-PAGE
gel showing samples of varying times of induction with IPTG in E. coli. Cells were induced in a
200mL flask with 0.1mM IPTG and placed in a 37C shaker for 30 minutes, 60 minutes, or 90
minutes. The E. coli cells were then lysed to obtain the samples.
Figure 1 shows previous induction of Rosetta
pLysS E. coli transformed with pET21
containing the Si-tag fusion protein was induced
with IPTG, cells were lysed, centrifuged, lysates
collected and analyzed by SDS-PAGE;
however, it the Si-tag fusion protein was
collected more in the protein rather than the
supernatant. Our goal is to get all of the protein
into the supernatant rather than from the pellet.
In figure 2, we were able to collect all the Si-tag
fusion protein in the supernatant. Thick bands
in Figure 2B indicate that the Si-tag fusion
protein was successfully lysed and collected in
the supernatant.
Ladder
0.5MMgCl2Sup
1MMgCl2Sup
1.5MMgCl2Sup
2MMgCl2Sup
.5MMgCl2Pellet
1MMgCl2Pellet
1.5MMgCl2Pellet
2MMgCl2Pellet
48
35
25
17
11
kDa
75
Figure 6. Purification using Si-tag’s affinity for Silica. Differing
concentrations of MgCl2 were used to find optimal eluting ability.