1. The structure of the scaffold for
cellular growth in-vitro is imperative
for proper cellular adhesion and
proliferation. Previous studies
introduced an electrospun,
mesoporous silica nanofiber (SNF) as
potential scaffold.1 The biodegrability
and biocompatibility of SNF makes it
an optimal scaffold for recombinant
fusion protein Sitag/RGD/His-tag.
Fusion Protein
Taniguchi et al discovered an E. coli
ribosomal L2 protein which has
strong affinity for silica, termed silica
binding protein (Si-tag).2 Attaching
Si-tag to a cell attachment domain
(RGD) as well as a purification
domain (His-tag) allows for the
building of fusion protein for the use
of tissue engineering on SNF.
Scaffold
Lysing is the process of breaking open a cell to extract what is needed on the
inside. There are many different methods to lysing cells; the main categories
include: mechanical, high frequency sound waves, freeze/thaw, chemical, and
liquid homogenization (Fig. 3). Different methods and lysing times were tested to
find the optimal condition to break open the cell and extract the most protein into
the supernatant.The methods tested for lysis include freeze thaw/B-per (Fig. 4), B-
per /lysozyme (Fig. 5), and TE (Tris and EDTA buffer )/lysozyme (Fig. 6). B-Per is a
detergent in a tris buffer. Detergent lysis solutions work by breaking open the lipid
barrier surrounding cells and disrupting interactions and bonds within the cell.
Figure 14. PC12 Cells Differentiating on Cover Slip, PLL, and SB Protein. Cells were exposed to
differentiation media and coated on three surfaces (A) Image of PC12 cells coated on plain
coverslip as a negative control; (B) cells coated on Poly-L-Lysine (PLL) as a positive control; (C)
2DIV and (D) 6DIV PC12 cells coated on Si-RGD-His tag fusion protein.
Cell/ PC12
Purification and Lysis of the Functional Si/RGD/His-tag Fusion Protein
Adam Hildebrandt, Anna Augustine, Samantha Brennan
Supervisor Dr. Mary Ann Yang
His-tag Purification
References
1. Chen WS, Hsieh PH, Yang WN, et al. Chemically modified electrospun silica
nanofibers for promoting growth and differentiation of neural stem cells. J Mater
Chem B. 2014;2:1205–1215.
2. Taniguchi K. 2006. The Si-Tag for Immobilizing Proteins on a Silica Surface. 96. 1023-
1029.
3. Polyhistidine-tag. (n.d.). Retrieved April 25, 2016, from
https://en.wikipedia.org/wiki/Polyhistidine-tag
4. 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 protein. 71. 91-95.
5. https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/traditional-methods-cell-lysis.html
6. Coyle B., Baneyx F. 2014. A Cleavable Silica-Binding Affinity Tag for Rapid and
Inexpensive Protein Purification. Biotechnology and Bioengineering. 111:10.
7. Ikeda T et al. The silica-binding Si-tag functions as an affinity tag even under
denaturing conditions. 2010. Protein Expression and Purificiation. 77: 173-177.
Lysis
A B
C D
Once lysing and purification is optimalized, the protein can be coated onto
glass coverslips. The protein then will allow PC12 cells to sit down on the
scaffold. Which in turn will allow the PC12 cells to grow and differentiated on
the coverslip.
Fig. 4. Purifcation of Sitag/RGD/His-tag fusion protein
utilizing His-tag’s affinity for Nickel.
Fig. 1. Construct of SNF, demonstrating
the three dimensional configuration
Fig 2. Construct of the Sitag/RGD/His-tag
fusion protein
Project Goals
This poster focuses on the experimental lysis and purification techniques that
were done to obtain the Sitag/RGD/His-tag fusion protein. Purified fusion
protein can then be used as a functional portion of the tissue engineering
scaffold.
Affinity tags, generally fused to the N- or C- terminus of expression targets are used to purify desired proteins.6 A
polyhistidine-tag (Histag) is an amino acid motif that consists of at least six histidine (His) residues and is used to
purify recombinant proteins expressed in E. coli.3 In combination with an IMAC column and an affinity resin, the
recombinant Sitag/RGD/His-tag fusion protein is able to be purified (Fig. 4). The resin contains high affinity
transition metal ions immobilized on a solid support through a coordinating ligand.6 Soluble cellular lysates are
added to the column, centrifuged, washed with a salt buffer, and eluted. Washing should remove all non-specific
cellular proteins. Elution, using imidazole, which is a high salt, elutes the protein by competing with binding spots
on the nickel resin. Subsequent to elutions, purified Sitag/RGD/His-tag fusion protein should be obtained.
In context of tissue engineering, it is imperative the Sitag/RGD/His-tag fusion protein is pure.
Any residual bacterial proteins deems the samples unusable for potential neural growth for
patients. Figure 5 see to the left shows the idea schematic for protein purification. Elution lanes
show no presence of residual protein. Figure 6 shows the initial results of purification. The
presence of residual proteins in the elution lanes led us to increase the number of washes, in
hopes to wash away more non-specific proteins. Figure 7 shows the results after increasing the
number of washes from 3 to 6. The presence of proteins still in the elution lanes even after the
increase in washes led to the change in induction concentration from 1.0 mM IPTG to 0.5 mM.
The thought was the higher IPTG concentration was causing protein to coagulate and therefore
caused the Sitag/RGD/His-tag fusion protein to be unable to bind with the nickel resin. The
result of poor yield, residual proteins, and expensive equipment led to moving to a different
purification method.
Fig. 7. Purification with increased washes to
six to rid non-specific proteins
Fig. 5. Ideal schematic of His-tag purification Fig. 6. SDS-PAGE gel of His-tag purification.
Fig. 8. His-tag purification using decreased IPTG
concentration.
Sitag Purification
The strong bindiing of silica is attributed to the unusual basicity, positive residues and disorderd structure of the Sitag
polypeptide.6 Sitag, an intrinsically disordered (ID) protein has fluctuating structures which allows for flexibility and optimal
binding to increase its intermolecular forces. Its positively charged side chain and residues (lysine) allow it to bind to the negatively
charged silica surfaces. Sitag also binds through its apolar side chains and the hydrophobic silica surface. Previous studies showed
that even under denaturing conditions (urea), Sitag remains bound, confirming that the binding is independent of structure.7 This
purification method utilizes silica nanoparticles and the stated Sitags affinity to them. Varying elution methods were tested.
Elution with MgCl2 Elution with L-lysine
Previous studies done by Ikeda showed that MgCl2 is able to elute
the fusion protein by interfering with Sitag’s binding to silica (Fig
9).4 It was found that divalent cations such as Mg2+ and Ca2+ could
release Sitag at a high concentration (2M); studies found however
that because Mg2+ has a higher ionic potential, it is more effective
than Ca2+. Due to the little Sitag released when using NaCl, it
suggests that divalent cations play an important role in
dissociating the protein.
Fig. 12. A) Shows the washes and elution flow-
throughs from purification while B) shows
concentrate and SNPs. The fusion protein was
unable to be dissociated from the SNPs. These
figures show the effects of different
concentrations of L-Lysine on dissociating the
Si-Tag fusion protein from silica nanoparticles.
Fig. 13. Increased washes and incubation time.
Fusion protein was able to be eluted form SNPs
via L-lysine.
Fig 10. There is high
levels of gel
disruption in this
figure due to high
amounts of salt
remaining in the
samples. It is
hypothesized that the
high salt
concentration
wouldn’t be optimal
for neuron
differentiation.
Future Work
Fig 9.
Results from
Ikeda
studies. Lane
1: control
Lane 5M
NaCl Lane 3:
2 M MgCl2
Lane 4: 2 M
CaCl2, Lane
5: 1 N HCl
and Lane 6:
1N NaOh
Divalent cations such as MgCl2 have a much higher ionic potential
than Na+ ions; therefore divalent cations are electrostatically
attracted to negatively charged silica surfaces more strongly than
monovalent Na+. This suggests that Sitag proteins are
electrostatically absorbed on silica surfaces and are then
dissociated by an ion exchange effect where Mg2+ ions act as
competition for the silanoal groups. 4 This competition allows for
the dissociation of the fusion protein.
Figure 3. Description of Lysing Methods. A table
explaining how different techniques of lysing work and
what is needed for each process.
Figure 4. SDS-PAGE Results from Lysing E.coli Cells using B-
Per/Lysozyme 1mg/mL and Dry Ice Freeze Thaw. SDS-PAGE
comparing lysing with dry ice and without. Different lysing times
of 30 and 60 minutes were also tested. All samples were lysed
with B-per/lysozyme 1mg/mL as well.
Figure 5. SDS-PAGE Showing Protein Sample Results
from Testing Different Lysing Conditions. Two different
conditions of TE/lysozyme buffer with final concentration
of 1mg/mL and B-per/DNase/ lysozyme buffer with a
1mg/mL final concentration were tested for optimal lysing
on Rosetta E.coli. All conditions were incubated at 37˚C
water. bath for either 30 or 60 minutes
Figure 6. SDS-PAGE Results From TE/Lysozyme V.s B-
Per/Lysozyme at Different Concentrations. Optimal cell lysis was
tested by comparing the effectiveness of increasing B-
Per/Lysozyme concentration from 200mg/mL to 1.0mg/mL.
These conditions were tested against TE/Lysozyme at 1.0mg/mL
final concentration. . All conditions were incubated at 37˚C water.
bath for 30 minutes.
If further testing isn’t indicative of Si-Tag purification; this
research might shift towards using a silica gel instead of
using silica nanoparticles. This method is proven to be
quickly purify the Car9 fusion protein.6 It is hypothesized
that the nanoparticles might instigate too strong of an
association for effective elution using L-Lysine. Silica gel is
what is used in.6 An apparatus will be assembled with two
plastic syringes with a valve connecting them (figure 15).
Silica gel is placed on top of glass wool and then is
immersed in wash buffer.6 Next, the cellular lysate can be
added to the system. As the suction pressure is being
applied, the fusion protein should theoretically bind to the
silica while all other unwanted items can flow through the
system by simply pulling the bottom syringe. To elute the
fusion protein from the apparatus, L-Lysine will be used to
elute the fusion protein to effectively purify it. The suction
pressure applied from the syringe at the bottom effectively
increases flow rate as well as purification time.6
Figure 15. Two syringe
system adapted from
(Coyle et al., 2014). This
system uses silica gel and
pressures from the syringe
to purify fusion proteins.
The first purification ran using L-lysine (Fig 10) shows that L-
lysine was unable to dissociate the protein. In efforts to find a
cheap and effective method for eluting the fusion protein from
silica, it was successfully proven that l-lysine effectively
dissociates GFPmut2-Car9 from silica.6 It is suggested in this
article, that the binding of the Car9 fusion protein involves
electrostatic interactions. Electrostatic interactions are
molecular associations that occur between basic and acidic
regions of molecules due to positive and negative electron
charges. In solution, the silica surface is converted in to silanol
groups; it is these silanol groups that interact with the basic
regions of the desired fusion protein.6 In addition to
electrostatic interactions, the association of the fusion protein
to silica also contains hydrophobic organization between the
central phenylalanine and siloxane groups.6 L-Lysine is a
positively charged amino acid that can compete for the silanol
binding sites through electrostatic interactions. Also, L-Lysine
contains an R group that is hydrophobic in nature. In tandem,
these properties theoretically allow for L-Lysine work to act as
an eluent for the Si-Tag fusion protein. It was discovered that a
1M solution of L-Lysine effectively eluted the Mut2-Car9 fusion
protein from silica with a purity of about 80%.6 The rational
behind testing L-Lysine elution is that it is a cheap, effective
and fast method for eluting fusion proteins.6 Coyle states that
using L-Lysine for elution costs two dollars per run in worst-
case scenarios. This is around ten times cheaper than a
previously used His-Tag purification. Also, purification of the
fusion protein could be obtained from cellular lysate in as little
as 15 minutes. The next logical step was to elute the Si-Tag
fusion protein from the silica nanoparticles using 1M L-Lysine.
Early results indicated that the interactions between the silica
nanoparticles and the Si-Tag fusion protein were too strong for
elution with 1M L-Lysine. Increased concentrations of L-Lysine
indicated some purification. Although some bacterial proteins
still remained.
Results indicated that B-PER acts as the best detergent (Fig. 5) when referencing
the bands for the supernatant. Freeze-thaw cycles showed no improvement for
lysis and was therefore not used in future experiments (Fig. 4). Increasing B-PER
concentration to 200 mg/ml showed no improvement in the recovered
supernatant fusion protein.
Figure 11. Adapted from (Coyle et al.,
2014) Si-Tag fusion protein association
with silanol groups located on the silica
surface
A
B