Summary [1]
Explanation about Parameters Tested by Us
Since we are a team that focus on the biosynthesis and application of
the Iron Oxide Nanoparticles, specifically the specific targeting of the
antibody linked Iron Oxide Nanoparticles to the HER2 positive cell,
following important things were tested by us:
1. Whether the bacteria strain chosen by us could properly synthesize the Iron Oxide Nanoparticles, and which magnetic state it would be in, what would the average diameter and polydispersity of the biologically synthesized nanoparticles be, whether the method we used to separate IONPs was good. This should be tested by TEM and DLS analysis after using the method we designed to separate the nanoparticles.
2. Whether the scFv domain of the anti-HER2 antibody could be properly expressed in the bacteria strain chosen by us, and whether the protein could be biologically active after purification (which means that binding with HER-2 positive cells should occur). This should be tested by immunostaining, flow cytometry analysis and western blot analysis by setting proper comparison groups and conduct the same staining to HER-2 negative cells.
3. Whether the biologically synthesized nanoparticles could be conjugated with the scFv domain of the anti-HER2 antibody effectively. This should be tested also by immunostaining if the second task could be proven. Or we could conduct the conjugation method on the chemically synthesized nanoparticles which shows significant cytotoxicity, and compare the result with unlinked groups to indirectly show both the biological activity of scFv synthesized by us and the success of the conjugation methods.
1. Whether the bacteria strain chosen by us could properly synthesize the Iron Oxide Nanoparticles, and which magnetic state it would be in, what would the average diameter and polydispersity of the biologically synthesized nanoparticles be, whether the method we used to separate IONPs was good. This should be tested by TEM and DLS analysis after using the method we designed to separate the nanoparticles.
2. Whether the scFv domain of the anti-HER2 antibody could be properly expressed in the bacteria strain chosen by us, and whether the protein could be biologically active after purification (which means that binding with HER-2 positive cells should occur). This should be tested by immunostaining, flow cytometry analysis and western blot analysis by setting proper comparison groups and conduct the same staining to HER-2 negative cells.
3. Whether the biologically synthesized nanoparticles could be conjugated with the scFv domain of the anti-HER2 antibody effectively. This should be tested also by immunostaining if the second task could be proven. Or we could conduct the conjugation method on the chemically synthesized nanoparticles which shows significant cytotoxicity, and compare the result with unlinked groups to indirectly show both the biological activity of scFv synthesized by us and the success of the conjugation methods.
Major Achievements
1. We successfully synthesized the Iron Oxide Nanoparticles (IONPs) E. coli
and the method used to separate IONPs are designed and tested by ourselves.
2. We successfully characterized the IONPs using TEM and DLS and found that using ok our method, the IONPs got by us were generally in superparamagnetic state and suitable for application need magnetic induction and won't let particles aggregrate.
3. We successfully synthesized the scFv domain of anti HER2 antibody in E. coli and purified the protein using His-tag purification column.
4. We showed that the biologically synthesized IONPs have no obvious cytotoxicity to both the HER2 positive cells and the HER2 negative cells, which might be caused by its low concentration. In comparison, the bare chemically synthesized IONPs show obvious cytotoxicity to both the HER2 positive cells and the HER2 negative cells. Also, the linkage of antibodies with scFv domain to the chemically synthesized IONPs could give them specificity against HER2 positive breast cancer cells. This indicates that the biologically synthesized nanoparticles are better for application if higher concentration and good specificity could be achieved in the future.
2. We successfully characterized the IONPs using TEM and DLS and found that using ok our method, the IONPs got by us were generally in superparamagnetic state and suitable for application need magnetic induction and won't let particles aggregrate.
3. We successfully synthesized the scFv domain of anti HER2 antibody in E. coli and purified the protein using His-tag purification column.
4. We showed that the biologically synthesized IONPs have no obvious cytotoxicity to both the HER2 positive cells and the HER2 negative cells, which might be caused by its low concentration. In comparison, the bare chemically synthesized IONPs show obvious cytotoxicity to both the HER2 positive cells and the HER2 negative cells. Also, the linkage of antibodies with scFv domain to the chemically synthesized IONPs could give them specificity against HER2 positive breast cancer cells. This indicates that the biologically synthesized nanoparticles are better for application if higher concentration and good specificity could be achieved in the future.
Approaches Without Success / Unexplained Confusion in Data We Got
1. We tried to use the immunostaining to examine the conjugation of the scFv domain
to the HER2 positive cell. But the result shows that the
anti-polyhis antibody
doesn't have much specificity to the primary antibody, indicating we couldn't
test the conjugation of the scFv domain to the HER2 positive cell using
this method or to say this antibody.
2. Now the binding affinity of the antibody designed by us could only be partially given from the cytotoxicity examination, which is not quite convincing.
3. In one of our DLS characterization, the hydrodynamic diameter of the nanoparticles were much greater than other repeats through exactly the same treatment, which is unexpected. And the only difference observed by us is that, only this time there did exist partially ropy stuff (like crude DNA extract) in the supernantant after centrifugation of the cell lysate. We are not sure whether this is directly related to the unusual hydrodynamic diameter.
One assumption of us is that since the viscosity of the sample we got that time is much larger, that would affect the speed of the brownian motion, thus affect the final hydrodynamic radius measured.
2. Now the binding affinity of the antibody designed by us could only be partially given from the cytotoxicity examination, which is not quite convincing.
3. In one of our DLS characterization, the hydrodynamic diameter of the nanoparticles were much greater than other repeats through exactly the same treatment, which is unexpected. And the only difference observed by us is that, only this time there did exist partially ropy stuff (like crude DNA extract) in the supernantant after centrifugation of the cell lysate. We are not sure whether this is directly related to the unusual hydrodynamic diameter.
One assumption of us is that since the viscosity of the sample we got that time is much larger, that would affect the speed of the brownian motion, thus affect the final hydrodynamic radius measured.
Future Work
1. As the existance of the biologically synthesized nanoparticles was proven,
we could further examine the coating composition by using Surface Plasmon Resonance or
Raman spectroscopy to see if the predicted composition or chemical groups
do exist on the membrane, this could further help us design new conjugation methods
towards both antibodies and drugs.
2. We could use the same conjugation method to further attach drugs like Doxorubicin to the biologically synthesized nanoparticles to see if the cytotoxicity could be increased, and further examine the specificity of the antibodies.
3. We could renew our antibody design by expanding the design to outside the scFv domain, as full structure of the antibody may improve the biological activity of the antibody. But since the supporter of protein expression chose by us was still bacteria, the proper folding of the whole protein could be unreliable. So we could express the protein in eukaryote cells like 293F. (one type of human embryonal kidney (HEK) cells)
4. We could further examine the conjugation of the scFv domain to the HER2 positive cell by attaching a fluorescent group to the scFv domain and directly stain the cell to see if the conjugation is successful.
2. We could use the same conjugation method to further attach drugs like Doxorubicin to the biologically synthesized nanoparticles to see if the cytotoxicity could be increased, and further examine the specificity of the antibodies.
3. We could renew our antibody design by expanding the design to outside the scFv domain, as full structure of the antibody may improve the biological activity of the antibody. But since the supporter of protein expression chose by us was still bacteria, the proper folding of the whole protein could be unreliable. So we could express the protein in eukaryote cells like 293F. (one type of human embryonal kidney (HEK) cells)
4. We could further examine the conjugation of the scFv domain to the HER2 positive cell by attaching a fluorescent group to the scFv domain and directly stain the cell to see if the conjugation is successful.
TEM Characterization of Biologically Synthesized Nanoparticles [2]
Method Description
In our examination,
Talos L120C G2 Transmission Electron Microscope was used for the characterization of the
nanoparticles.
Basically, this microscope utilizes a beam of electrons instead of light to visualize the sample, enabling incredibly detailed images with a resolution down to the sub-nanometer scale.
To begin the analysis, we prepare a sample of the nanoparticles by depositing a thin layer onto a carbon-coated copper grid. This grid is carefully loaded into the Talos L120C G2 TEM, ensuring proper positioning for imaging and analysis.
Once the sample is inserted, we set the imaging parameters according to our specific requirements. The TEM employs a high-energy electron beam that is directed onto the sample. As the electrons interact with the nanoparticles, they scatter and interact with the internal structure of the particles.
We observe and capture the scattering patterns produced by the interaction of the electrons with the nanoparticles. These patterns are collected by a detector and transformed into high-resolution images, providing us with detailed information about the morphology, size, and distribution of the nanoparticles.
In addition to imaging, we also utilize Selected Area Electron Diffraction (SAED) analysis with the Talos L120C G2 TEM. By selecting a specific area of interest on the sample using an aperture, we focus the electron beam onto that region. The resulting diffraction pattern reveals the crystalline properties and orientation of the nanoparticles, enabling us to determine their structural characteristics.
They provide us with valuable information about the size, shape, and distribution of the nanoparticles, as well as give us one way to compare the sample with uninduced control to show the success of the synthetic process.
Basically, this microscope utilizes a beam of electrons instead of light to visualize the sample, enabling incredibly detailed images with a resolution down to the sub-nanometer scale.
To begin the analysis, we prepare a sample of the nanoparticles by depositing a thin layer onto a carbon-coated copper grid. This grid is carefully loaded into the Talos L120C G2 TEM, ensuring proper positioning for imaging and analysis.
Once the sample is inserted, we set the imaging parameters according to our specific requirements. The TEM employs a high-energy electron beam that is directed onto the sample. As the electrons interact with the nanoparticles, they scatter and interact with the internal structure of the particles.
We observe and capture the scattering patterns produced by the interaction of the electrons with the nanoparticles. These patterns are collected by a detector and transformed into high-resolution images, providing us with detailed information about the morphology, size, and distribution of the nanoparticles.
In addition to imaging, we also utilize Selected Area Electron Diffraction (SAED) analysis with the Talos L120C G2 TEM. By selecting a specific area of interest on the sample using an aperture, we focus the electron beam onto that region. The resulting diffraction pattern reveals the crystalline properties and orientation of the nanoparticles, enabling us to determine their structural characteristics.
They provide us with valuable information about the size, shape, and distribution of the nanoparticles, as well as give us one way to compare the sample with uninduced control to show the success of the synthetic process.
Results / Data of 5 mM FeCl3 Induced Culture
The representative TEM figures of the biologically synthesized nanoparticles and the negative control with
no ferric ion induction are shown below.
To get the size distribution of the nanoparticles, we use ImageJ to analyze the TEM images.
Multiple figures were chosen for data processing. In total fifty to seventy particles were randomly selected
in each figure,
the size distribution statistical chart of the nanoparticles is shown below.
Figure 1. Biologically Synthesized Nanoparticle by 5 mM FeCl3 Induction Attached to Biomass.
Figure 2. Biologically Synthesized Nanoparticle by 5 mM FeCl3 Induction Detached from Biomass through ultrasonication.
Figure 3. Negative Control for Nanoparticle without Ferric Ion Induction.
Figure 4. Size Distribution of the Biologically Synthesized Nanoparticles.
From the size distribution statistical data, the Polydispersity Index of the samples in each view field was calculated and shown as below:
Figure 5. Polydispersity Index of the Samples in Each View Field.
All the figures used for data processing are labelled and shown in the supplementary data section below.
Analysis / Discussion
Firstly, the comparison between ferric induced culture and uninduced culture shows the success of the
biological
synthesis of the nanoparticles. There are no nanoparticles observed in the uninduced culture, (the faint
grain in
the TEM figures for uninduced culture were actually some inorganic salt as when electron-beams were fired
to the sample,
the salt grain would disappear), while the induced culture shows a large amount of nanoparticles
attached to the biomass or off the biomass.
Secondly, the size distribution of the nanoparticles shows that the nanoparticles are in the range of 5-10 nm, this means that almost all the nanoparticles synthesized by us are in superparamagnetic state, indicating the applicability of biologically synthesized nanoparticle in tumor penetration or tumor accumulation.
Thirdly, the comparison between the sample attached to the biomass and the sample detached from the biomass shows that in separation of the biological synthesized nanoparticles, ultrasonication chosen by us is a proper method to detach the nanoparticles from the biomass.
Fourthly, the Polydispersity Index of the samples in each view field shows that the nanoparticles are relatively uniform in size.
Secondly, the size distribution of the nanoparticles shows that the nanoparticles are in the range of 5-10 nm, this means that almost all the nanoparticles synthesized by us are in superparamagnetic state, indicating the applicability of biologically synthesized nanoparticle in tumor penetration or tumor accumulation.
Thirdly, the comparison between the sample attached to the biomass and the sample detached from the biomass shows that in separation of the biological synthesized nanoparticles, ultrasonication chosen by us is a proper method to detach the nanoparticles from the biomass.
Fourthly, the Polydispersity Index of the samples in each view field shows that the nanoparticles are relatively uniform in size.
Selective Electron Diffraction Results and Analysis
Beyond simply observing the morphology of the nanoparticles, we also utilize
Selected Electron Diffraction analysis to gain a deeper understanding of their
structural properties.
The raw data of the Selected Electron Diffraction analysis of the biologically synthesized nanoparticle was shown below:
The result shows that the sample is a type of polycrystal with 6 crystal faces. The crystal faces from inner to the outer are numbered 1 to 6. The distance between each layer is 3.6nm, 1.03nm, 0.42nm, 0.65nm, 0.36nm. It means that the samples don't have a uniform structure and the specific crystal type of our sample can't be further analyzed. Maybe it is because there still remains some biological structures attached on our samples which corresponds to the layer 3,4,5,6 on the picture. More accurate separation process could be done to further separate the unwanted biological structures. In all it proved the existance of crystal structure in our samples.
The raw data of the Selected Electron Diffraction analysis of the biologically synthesized nanoparticle was shown below:
Figure 6. Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticles Taken in 8/23
Figure 7. Selected Electron Diffraction Pattern of Biologically Synthesized Nanoparticles Taken in 8/25
The result shows that the sample is a type of polycrystal with 6 crystal faces. The crystal faces from inner to the outer are numbered 1 to 6. The distance between each layer is 3.6nm, 1.03nm, 0.42nm, 0.65nm, 0.36nm. It means that the samples don't have a uniform structure and the specific crystal type of our sample can't be further analyzed. Maybe it is because there still remains some biological structures attached on our samples which corresponds to the layer 3,4,5,6 on the picture. More accurate separation process could be done to further separate the unwanted biological structures. In all it proved the existance of crystal structure in our samples.
Supplementary Datas
The supplementary figures and statistical information of the TEM characterization of the biologically
synthesized nanoparticles could be found in the pdf file below.
TEM Characterization of Biologically Synthesized
Nanoparticles and Size Distribution Statistics
DLS and Zeta Potential Characterization of Biologically Synthesized Nanoparticles and Zeta Potential Measurement [3]
Method Description
For the DLS characterization and Zeta Potential measurement of the biologically synthesized
nanoparticles, we use
Brookhaven NanoBrook
Series.
This advanced instrument utilizes Dynamic Light Scattering (DLS) technology, which allows us to measure the size distribution of nanoparticles in a liquid suspension. By analyzing the fluctuations in the intensity of scattered light caused by the random motion of nanoparticles, we can obtain valuable information about their size, size distribution, and polydispersity.
When it comes to analyzing our biologically synthesized nanoparticles, the NanoBrook Series offers a user-friendly and efficient workflow. Firstly, we prepare a sample of our nanoparticles in a suitable liquid medium, ensuring that the sample is well-dispersed. Then we introduce the sample into the NanoBrook instrument, where it is carefully illuminated with a laser beam.
The scattered light from the nanoparticles is collected and analyzed, allowing us to obtain valuable data on the size distribution of the particles. This information is crucial for evaluating the quality of our synthesized nanoparticles and understanding their behavior in biological systems.
Besides, the NanoBrook Series enables us to measure the Zeta Potential of our nanoparticles, \ which is a key parameter that provides insights into the stability and surface charge of nanoparticles. Also, by assessing the electric potential at the particle's surface, we can gain a deeper understanding of their interactions with their environment.
This advanced instrument utilizes Dynamic Light Scattering (DLS) technology, which allows us to measure the size distribution of nanoparticles in a liquid suspension. By analyzing the fluctuations in the intensity of scattered light caused by the random motion of nanoparticles, we can obtain valuable information about their size, size distribution, and polydispersity.
When it comes to analyzing our biologically synthesized nanoparticles, the NanoBrook Series offers a user-friendly and efficient workflow. Firstly, we prepare a sample of our nanoparticles in a suitable liquid medium, ensuring that the sample is well-dispersed. Then we introduce the sample into the NanoBrook instrument, where it is carefully illuminated with a laser beam.
The scattered light from the nanoparticles is collected and analyzed, allowing us to obtain valuable data on the size distribution of the particles. This information is crucial for evaluating the quality of our synthesized nanoparticles and understanding their behavior in biological systems.
Besides, the NanoBrook Series enables us to measure the Zeta Potential of our nanoparticles, \ which is a key parameter that provides insights into the stability and surface charge of nanoparticles. Also, by assessing the electric potential at the particle's surface, we can gain a deeper understanding of their interactions with their environment.
Results / Data of 5 mM FeCl3 Induced Culture
DLS Analysis
The size distribution pattern of the biologically synthesized nanoparticles using log scale is shown below:
Zeta Potential Measurement
For biologically synthesized nanoparticles, the Zeta Potential measured by us shows an average of -21.52 mV, which is larger than the chemically synthesized nanoparticles in its absolute value .
Raw data of the Zeta Potential measurement of the biologically synthesized nanoparticles is shown below:
As a comparision, for the chemically synthesized nanoparticle, the Zeta Potential measured by us shows an average of -20.26 mV, which is smaller than the biologically synthesized nanoparticles in its absolute value.
Raw data of the Zeta Potential measurement of the chemically synthesized nanoparticles is shown below:
The size distribution pattern of the biologically synthesized nanoparticles using log scale is shown below:
Figure 1. Biologically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.
From the size distribution pattern, the average hydrodynamic diameter of nanoparticles is 7.23 nm.
The size distribution pattern of the chemically synthesized nanoparticle using log scale is
shown below:
Figure 2. Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.
From the size distribution pattern, the average hydrodynamic diameter of nanoparticles is 252.41 nm.
Actually the real size of the chemically synthesized nanoparticles by TEM analysis is around 20 nm,
but
the
hydrodynamic diameter of the nanoparticles sythesized by us is much larger than their real size due to the
aggregration of the nanoparticles,
this also indicates that the chemically synthesized nanoparticles
are not that stable and are easier to get precipitated out.
For clearer representation, the linear scale size distribution pattern of the chemically synthesized
nanoparticles is shown below:
Figure 3. Chemically Synthesized Nanoparticle Size Distribution Pattern by DLS Analysis.
For biologically synthesized nanoparticles, the Zeta Potential measured by us shows an average of -21.52 mV, which is larger than the chemically synthesized nanoparticles in its absolute value .
Raw data of the Zeta Potential measurement of the biologically synthesized nanoparticles is shown below:
Figure 4. Zeta Potential Measurement of the Biologically Synthesized Nanoparticles.
And the supplementary all field data of the Zeta Potential measurement of the biologically synthesized nanoparticles is shown in the pdf file below:Zeta Potential Measurement of the Biologically Synthesized
Nanoparticles
As a comparision, for the chemically synthesized nanoparticle, the Zeta Potential measured by us shows an average of -20.26 mV, which is smaller than the biologically synthesized nanoparticles in its absolute value.
Raw data of the Zeta Potential measurement of the chemically synthesized nanoparticles is shown below:
Figure 5. Zeta Potential Measurement of the Chemically Synthesized Nanoparticles.
And the supplementary all field data of the Zeta Potential measurement of the chemically synthesized nanoparticles is shown in the pdf file below:Zeta Potential Measurement of the Chemically Synthesized
Nanoparticles
Analysis / Discussion
Firstly, for the DLS result of biologically synthesized nanoparticles, the results give the
average hydrodynamic diameter to be 7.23 nm, which is in superparamagnetic state and not
easy to form aggregation. This is consistent with the result of TEM analysis.
While for the chemically synthesized nanoparticles, the results give the average hydrodynamic diameter to be 252.41 nm, which is much larger than the real size of the nanoparticles, indicating that the nanoparticles are easier to form aggregation and not easy to get separated by untrasonic treatment. This is also consistent with the result of TEM analysis.
Though here we only have one biologically synthesized nanoparticle sample tested, the consistent result of three samples from 10/2 showed good reproducibility of the biological synthesis process, separation method and feasibility of the synthetic process.
Secondly, for the Zeta Potential measurement of the biologically synthesized nanoparticles, the average Zeta Potential is -21.52 mV, in absolute value larger than the zeta potential of chemically synthesized nanoparticles.
As the Zeta Potential is a parameter that indicates the stability of the nanoparticles, the larger absolute value of the Zeta Potential of the biologically synthesized nanoparticles indicates that the biologically synthesized nanoparticles are more stable than the chemically synthesized nanoparticles. This result was also shown good reproductbility by comparing with results in the other days.
While for the chemically synthesized nanoparticles, the results give the average hydrodynamic diameter to be 252.41 nm, which is much larger than the real size of the nanoparticles, indicating that the nanoparticles are easier to form aggregation and not easy to get separated by untrasonic treatment. This is also consistent with the result of TEM analysis.
Though here we only have one biologically synthesized nanoparticle sample tested, the consistent result of three samples from 10/2 showed good reproducibility of the biological synthesis process, separation method and feasibility of the synthetic process.
Secondly, for the Zeta Potential measurement of the biologically synthesized nanoparticles, the average Zeta Potential is -21.52 mV, in absolute value larger than the zeta potential of chemically synthesized nanoparticles.
As the Zeta Potential is a parameter that indicates the stability of the nanoparticles, the larger absolute value of the Zeta Potential of the biologically synthesized nanoparticles indicates that the biologically synthesized nanoparticles are more stable than the chemically synthesized nanoparticles. This result was also shown good reproductbility by comparing with results in the other days.
Characterization of Nanoparticles Synthesized by Different Times and in different Ferric Ion Induction Concentrations [4]
Description
To optimize the large scale biological nanoparticles synthesis, we tested the effect of
different ferric ion induction concentrations and different induction time on the nanoparticles synthesis.
Three time gradient including 24 hours, 36 hours and 48 hours were tested, and four ferric ion
induction concentration gradient including 0 mM, 2 mM, 5 mM and 7.5 mM were tested. These designs came from
our preliminary test that iron concentration over 10 mM would greatly inhibit the growth of the bacteria
and its protein expression level.
For each group, the general processes were controlled to be the same as the protocol written in our experiment page. And characterizations were conducted by TEM and DLS analysis as introduced above.
For each group, the general processes were controlled to be the same as the protocol written in our experiment page. And characterizations were conducted by TEM and DLS analysis as introduced above.
Iron Concentration Gradient
By DLS and Zeta Potential measurement, we found the optimized concentration of ferric ion for nanoparticle
synthesis is 2.5 mM,
as no significant difference in average hydrodynamic diameter and polydispersity index was found between 2.5
mM
and 5 mM induced group but the absolute value of zeta potential of 2.5 mM induced group is
higher than 5 mM induced group, indicating a higher stability. Summarized data were shown in the following
figures:
Figure 1. Hydrodynamic diameter summary figure
Figure 2. Polydispersity index summary figure
Figure 3. Zeta potential summary figure
Time gradient
Here as the Concentrations of the samples were too low, we failed to get reliable zeta potential
data.
And the hydrodynamic diameter and polydispersity index data were shown below:
Here we could see that the diameter of the IONPs varies according to different induction time,
fulfilling
different requirements for different usage. And as for specific targeting with
the requirement of superparamagnetism, 24 hours induced group is the best choice.
Figure 4. Hydrodynamic diameter summary figure
Figure 5. Polydispersity index summary figure
Analysis
Unfortunately, the vacuum facilities of the TEM machine in our school was broken
during the time we were doing the final characterization for iron concentration gradient
and time gradient, so we could not get the TEM images for these two groups.
For the ferric ion gradient examination, the hydrodynamic diameter of all groups were larger than the expected data and the former data we got. As said in the summary above, there were one unusual phenomenon that after the centrifugation of the cell lysate, there were some ropy stuff (like crude DNA extract) in the supernantant. Since the viscosity of this is much larger than water, would affect the speed of the brownian motion.
But the result of the zeta potential measurement still show significant difference between samples with negative control and samples with ferric ion induction, indicating success of the synthesis.
And as all four samples shown similar phenomenon when conducting nanoparticle separation, and the negative control for 0 mM ferric ion induction group have zeta potential of nearly zero, such that the trend could still give a preliminary result that 2.5 mM is the optimized concentration for nanoparticle synthesis as the zeta potential and polydispersity of 2.5 mM induced group is the lowest among all the induced groups.
For the ferric ion gradient examination, the hydrodynamic diameter of all groups were larger than the expected data and the former data we got. As said in the summary above, there were one unusual phenomenon that after the centrifugation of the cell lysate, there were some ropy stuff (like crude DNA extract) in the supernantant. Since the viscosity of this is much larger than water, would affect the speed of the brownian motion.
But the result of the zeta potential measurement still show significant difference between samples with negative control and samples with ferric ion induction, indicating success of the synthesis.
And as all four samples shown similar phenomenon when conducting nanoparticle separation, and the negative control for 0 mM ferric ion induction group have zeta potential of nearly zero, such that the trend could still give a preliminary result that 2.5 mM is the optimized concentration for nanoparticle synthesis as the zeta potential and polydispersity of 2.5 mM induced group is the lowest among all the induced groups.
Examination of the Conjugation with scFv Domain to the HER2 Positive Cell [5]
Method Description
Basically, we used the immunostainning to examine the conjugation of the scFv domain to the HER2
positive cell.
As we only synthesized the scFv domain of the anti-HER2 antibody with his-tag attached to the end, we could not directly find a secondary antibody with fluorescent tag to bind to the scFv domain. Therefore, we use the mouse derived anti-polyhis antibody to attach the scFv domain synthesized by us and then use the tertiary goat derived anti-mouse antibody to label the anti-polyhis antibody.
For convenience of explanation, here we called the scFv domain to be the primary antibody, the anti-polyhis antibody to be the secondary antibody and the anti-mouse antibody to be the tertiary antibody.
Generally, to test the conjugation, we added these three antibodies to the HER2 positive cell and HER2 negative cell. And to see if the secondary antibody has relatively good specificity to the primary antibody, we also added one group that only had the secondary antibody and the tertiary antibody to the HER2 positive cell and HER2 negative cell. Also, the background and unstained fluorescent signal were also tested by two groups, one is only added the primary and secondary antibody to the HER2 positive cell and HER2 negative cell, the other is only added the secondary and tertiary antibody to the HER2 positive cell and HER2 negative cell.
Final results were tested by the confocal microscopy and flow cytometry analysis to see the mean fluorescence intensity of the cells.
We have the detailed design of the test as below:
As we only synthesized the scFv domain of the anti-HER2 antibody with his-tag attached to the end, we could not directly find a secondary antibody with fluorescent tag to bind to the scFv domain. Therefore, we use the mouse derived anti-polyhis antibody to attach the scFv domain synthesized by us and then use the tertiary goat derived anti-mouse antibody to label the anti-polyhis antibody.
For convenience of explanation, here we called the scFv domain to be the primary antibody, the anti-polyhis antibody to be the secondary antibody and the anti-mouse antibody to be the tertiary antibody.
Generally, to test the conjugation, we added these three antibodies to the HER2 positive cell and HER2 negative cell. And to see if the secondary antibody has relatively good specificity to the primary antibody, we also added one group that only had the secondary antibody and the tertiary antibody to the HER2 positive cell and HER2 negative cell. Also, the background and unstained fluorescent signal were also tested by two groups, one is only added the primary and secondary antibody to the HER2 positive cell and HER2 negative cell, the other is only added the secondary and tertiary antibody to the HER2 positive cell and HER2 negative cell.
Final results were tested by the confocal microscopy and flow cytometry analysis to see the mean fluorescence intensity of the cells.
We have the detailed design of the test as below:
| Cell Strain | Treatment of the cells | ||
|---|---|---|---|
| Anti HER2 scFv domain antibody | Anti His tag antibody (mouse derived) | Goat Anti-Mouse-IgG | |
| SK-BR-3 treatment 1 (three replica) |
- | + | + |
| SK-BR-3 treatment 2 (three replica) |
+ | + | + |
| SK-BR-3 treatment 3 (three replica) |
+ | + | - |
| SK-BR-3 treatment 4 (three replica) |
+ | - | + |
| MDA-MB-231 treatment 1 (four replica) |
- | + | + |
| MDA-MB-231 treatment 2 (three replica) |
+ | + | + |
| MDA-MB-231 treatment 3 (two replica) |
+ | + | - |
| MDA-MB-231 treatment 4 (two replica) |
+ | - | + |
Result
Sadly, the results of the confocal microscopy and flow cytometry analysis show that the
secondary
antibody don't have much specificity to the primary antibody, indicating we couldn't test the
conjugation of
the scFv domain to the HER2 positive cell using this method. The group only added the
secondary and
tertiary antibody also shows strong fluorescent signal, which should not occur if the secondary
antibody is a
good one.
The mean fluorescent intensity of the SK-BR3 under different treatments are shown below:
Note that as the background comparision groups were not included in the figures as the
effective
counted area were over 100 times smaller than positive groups, this way the mean fluorescent
intensity is not useful.
To further show the success of negative result, the figures of the confocal microscopy result after setting the photo to 8 bit and auto the threshold are shown below:
The figure shows that the background signal is weak, indicating the background fluorescence is weak.
The figure shows that the background signal is weak, indicating the specificity of tertiary antibody is high and was washed thoroughly.
The figure shows no obvious difference between the group adding all three antibodies and the group only adding secondary and tertiary antibodies, while two negative controls show normal results.
The mean fluorescent intensity of the SK-BR3 under different treatments are shown below:
Figure 1. Mean Fluorescent Intensity of the SK-BR3 under Different Treatments.
To further show the success of negative result, the figures of the confocal microscopy result after setting the photo to 8 bit and auto the threshold are shown below:
Figure 2. Confocal Microscopy Result of the SK-BR3 under Primary and Secondary Antibody Treatment.
The figure shows that the background signal is weak, indicating the background fluorescence is weak.
And the corresponding figure under bright field is shown below:
Figure 3. Bright Field Channel Result of the SK-BR3 under Primary and Secondary Antibody Treatment.
Figure 4. Confocal Microscopy Result of the SK-BR3 under Primary and Tertiary Antibody Treatment.
The figure shows that the background signal is weak, indicating the specificity of tertiary antibody is high and was washed thoroughly.
And the corresponding figure under bright field is shown below:
Figure 5. Bright Field Channel Result of the SK-BR3 under Primary and Tertiary Antibody Treatment.
The flow cytometry results also support the conclusion that the secondary antibody didn't show specificity towards the scFv domain.
Figure 6. Flow Cytometry Result of the SK-BR3 under Different Treatment.
The figure shows no obvious difference between the group adding all three antibodies and the group only adding secondary and tertiary antibodies, while two negative controls show normal results.
Method Two by Western Blot Analysis
Besides, we conducted western blot test to see if the scFv domain could bind to the
HER2 protein.
This was done by firstly adding the scFv domain to the cell well for 12-24 hours,
then washing the
well with PBS several times to remove the unbound scFv domain, then extracting the
protein for electrophoresis,
membrane transformation and staining. Comparison groups were the HER2 positive cell,
HER2 negative cell and empty well. Possible drawback of this design is that since
western
blot analysis required proper amount of protein content, the amount of scFv domain
added to the
cell plate may not be enough to show the band in the western blot analysis. We added
180 μL scFv-cys-polyhis
with 4.236 μg/μL concentration to the cell plate, which is 762 μg scFv-cys-polyhis
in total. But since we are
adding the protein into 10 cm cell dish, this amount is still not large enough.
Still the result showed no obvious band comparing to the positive control group (which have 20.544 μg protein in total by BCA test) in the western blot analysis, indicating two possibilities:
1. The scFv domain didn't bind to the HER2 protein.
2. The amount of scFv domain added to the cell plate is not enough to show the band in the western blot analysis.
The result of the western blot analysis is shown below:
Still the result showed no obvious band comparing to the positive control group (which have 20.544 μg protein in total by BCA test) in the western blot analysis, indicating two possibilities:
1. The scFv domain didn't bind to the HER2 protein.
2. The amount of scFv domain added to the cell plate is not enough to show the band in the western blot analysis.
The result of the western blot analysis is shown below:
Figure 7. Western Blot Analysis Result.
Future Plan
Though we failed to show the binding affinity of the scFv domain to the HER2
positive cell directly before the due day of this project, still we thought about
the method
to test the conjugation of the scFv domain to the HER2 positive cell.
As in our design, there have one type of scFv domain with a cysteine along with a His-tag attached to the end of the scFv domain, indicating that we could make use of the thiol group for conjugation with a fluorescent tag with maleimide. In this way we don't need to use the secondary and tertiary antibody here. Or we could use other common method used to link a fluorescent tag to a protein, such as directly link with the amine group on the scFv domain, using biotin-streptavidin system or click chemistry.
As for the improvement of the western blot analysis design, we could add more scFv domain and add less positive control scFv protein to improve the contrast. And more control groups like cells without treated with scFv domain should be added to show the specificity of the binding.
Also, we could change the design for the protein synthesized in the bacteria to the full length anti-HER2 antibody, which would make it easier to find proper secondary antibody to test the biological activity of it. But this would increase the risk of facing the problem of improper folding in bacteria. So we think the first or the second method is more convenient for future examination.
As in our design, there have one type of scFv domain with a cysteine along with a His-tag attached to the end of the scFv domain, indicating that we could make use of the thiol group for conjugation with a fluorescent tag with maleimide. In this way we don't need to use the secondary and tertiary antibody here. Or we could use other common method used to link a fluorescent tag to a protein, such as directly link with the amine group on the scFv domain, using biotin-streptavidin system or click chemistry.
As for the improvement of the western blot analysis design, we could add more scFv domain and add less positive control scFv protein to improve the contrast. And more control groups like cells without treated with scFv domain should be added to show the specificity of the binding.
Also, we could change the design for the protein synthesized in the bacteria to the full length anti-HER2 antibody, which would make it easier to find proper secondary antibody to test the biological activity of it. But this would increase the risk of facing the problem of improper folding in bacteria. So we think the first or the second method is more convenient for future examination.
Cytotoxicity Examination of the Biologically Synthesized Nanoparticles and Chemically Synthesized Nanoparticle [6]
Method Description / Experimental Design
The Cell Counting Kit-8 (CCK-8) is commonly used to assess cell viability and cytotoxicity, in which WST-8
(a yellow, water-soluble tetrazolium salt) is added to the cell culture and this tetrazolium salt is taken
up by live cells.
Inside metabolically active cells, mitochondrial dehydrogenases convert WST-8 into a water-soluble formazan dye. This conversion occurs in proportion to the metabolic activity of the cells. And the formazan dye produced is soluble in the cell culture medium and has an orange color. The intensity of the orange color is directly proportional to the number of living cells and their metabolic activity.
Therefore, it can be quantified by measuring the absorbance of the solution at a specific wavelength using a spectrophotometer or a microplate reader. In general, higher absorbance values indicate more viable and metabolically active cells, while lower values suggest reduced cell viability. Additionally, the calculation of the cell viability or cytotoxicity as a percentage use the following formula:
Cell Viability (%) = [(OD of treated sample - OD of blank control) / (OD of control sample - OD of blank control)] x 100
Therefore, here we used the cck-8 kit to test the cytotoxicity of IONPS to detect its cancer cells killing capacity, which represents therapeutic effects. And also, since we designed antibodies targeting the HER2 domain of breast cancer cells (bccs) and linked them to the IONPS to equip IONPS with specificity of killing bccs, the cytotoxicity differences between the IONPS linked to different antibodies and bare IONPS can help to verify the success of linkage of our biosynthesized antibodies to the IONPS and targeting effects of the antibodies.
We prepared samples of different concentrations in cell culture medium corresponding to the both the HER2 positive breast cancer cells BT-474 and HER2 negative breast cancer cells MDA-MB-231. And after thawing cells in 96-wells and the addition of the toxic substances (the samples of IONPs). The OD values were measured in a 96-plate reader. The measured OD values were then calculated by the formula given in the protocol of the cck-8 kit.
However, the first round of experiments indicated limited cytotoxicity of biosynthesized IONPs, which might be caused by its low concentrations, while chemically synthesized IONPS showed relatively obvious therapeutic effects. As a result, we conducted the following experiments mainly to verify therapeutic effects of the latter and the targeting effects of our antibodies.
Inside metabolically active cells, mitochondrial dehydrogenases convert WST-8 into a water-soluble formazan dye. This conversion occurs in proportion to the metabolic activity of the cells. And the formazan dye produced is soluble in the cell culture medium and has an orange color. The intensity of the orange color is directly proportional to the number of living cells and their metabolic activity.
Therefore, it can be quantified by measuring the absorbance of the solution at a specific wavelength using a spectrophotometer or a microplate reader. In general, higher absorbance values indicate more viable and metabolically active cells, while lower values suggest reduced cell viability. Additionally, the calculation of the cell viability or cytotoxicity as a percentage use the following formula:
Cell Viability (%) = [(OD of treated sample - OD of blank control) / (OD of control sample - OD of blank control)] x 100
Therefore, here we used the cck-8 kit to test the cytotoxicity of IONPS to detect its cancer cells killing capacity, which represents therapeutic effects. And also, since we designed antibodies targeting the HER2 domain of breast cancer cells (bccs) and linked them to the IONPS to equip IONPS with specificity of killing bccs, the cytotoxicity differences between the IONPS linked to different antibodies and bare IONPS can help to verify the success of linkage of our biosynthesized antibodies to the IONPS and targeting effects of the antibodies.
We prepared samples of different concentrations in cell culture medium corresponding to the both the HER2 positive breast cancer cells BT-474 and HER2 negative breast cancer cells MDA-MB-231. And after thawing cells in 96-wells and the addition of the toxic substances (the samples of IONPs). The OD values were measured in a 96-plate reader. The measured OD values were then calculated by the formula given in the protocol of the cck-8 kit.
However, the first round of experiments indicated limited cytotoxicity of biosynthesized IONPs, which might be caused by its low concentrations, while chemically synthesized IONPS showed relatively obvious therapeutic effects. As a result, we conducted the following experiments mainly to verify therapeutic effects of the latter and the targeting effects of our antibodies.
Result and Analysis
Generally, the results turned out that the cell viability showed negative correlation with increased
concentration of chemically synthesized IONPs (cIONPs) with antibodies and the corresponding bare IONPS.
But no evident cytotoxicity was shown for samples of bio-synthesized IONPs, which might be due to the low
concentration. Also, cIONPs linked with antibodies by T1 showed weak targeting specificity since there
shown limited differences between the cell viability of HER2 positive and HER2 negative cells. And cIONPs
linked with antibodies by T2 exhibited relatively evident targeting specificity on HER2 positive breast
cancer cells.
Note that Bio-AB represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2
antibody with poly-his tag attached at the end.
Chem-AB represents Chemically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody with poly-his tag attached at the end.
Bio-CH represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.
CH represents scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.
AB represents scFv domain of anti HER2 antibody with poly-his tag attached at the end.
For treatment 1 nanoparticles, the first figure of the test with the her-2 negative cells MDA-MB-231, all the groups exhibited obvious cytotoxicity for the cell viability of all groups showed low. And there did not exist considerate difference between control, bio-unlinked. Bio-AB, and bio-CH, as the cell viability were all relatively closed to 1.0.
And for her-2 positive cell BT-474 in the second figure, there did not show the obvious difference except chem-AB and chem-CH. The cell viability of adding chem-AB and chem-CH were lower than other groups significantly.
Compared two diagrams, there was no obvious change in cell viability in the two groups with the addition of chem-AB, which was convinced the cytotoxicity of the chem-AB. While the targeting effects of chem-CH were exhibited, as there did exist a obvious difference between positive cell BT-474 and negative cell MDA-MB-231 with cell viability in figure 1 was significantly higher than the figure 2.
Note that AB represents the anti HER2 antibody with poly his tag attached at the end and CH
represents anti HER2 antibody with poly-his tad attached at the end and here they were bound to some
of the nanoparticles with its scFv domain.
Chem_nps_1 and chem_nps_2 represent chemically synthesized nanoparticles with different batches.
For treatment 1 nanoparticles, the first figure of the test with the her2 positive cells BT-474, all the nps exhibited obvious cytotoxicity since the cell viability showed evident decline as the concentration of nps increased, which indicates the cytotoxicity of all three samples in T1 to be concentration-dependent. These nps showed a relevantly large cytotoxicity (close to 1) at low concentrations. And there did not exist considerate differences between cytotoxicity of nps-2 linked to antibodies and bare nps-2. Also, for her2 negative cells MDA-MB-231 in the second figure, all three nps showed similar effects of lowering the cell viability with increased concentrations. These results can be roughly concluded that the nps have great killing effects on these two breast cancer cells and the linkage of antibodies by treatment 1 could have failed to equip our chemical synthesized nps with expected specificity to her2 positive cells, which means the targeting effects of the anti-her2 antibodies do not seem to function after linkage.
Note that AB here represents the same antibodies as in treatment 1 and CH represents anti HER2
antibody with poly-his tad attached at the end.
For treatment 2 nanoparticles, figure 1 shows the killing effects of nps on the her2 positive cells BT-474. The cell viability showed mild decline as the concentration of nps with antibodies AB and CH increased, which indicates the cytotoxicity of all three samples in T2 to be concentration-dependent as well. And the curve of cell viability of bare antibodies here does not show evident trend, differing from nps linked to antibodies. Since the cytotoxicity of chemically synthesized nps has been proved in previous experiments, this result indicates nps linked to antibodies successfully targeted HER2 positive breast cancer cells. The subsequent tests conducted on HER2 negative breat cancer cells MDA-MB-231 (in figure2) showed limited tendency of concentration-dependent cytotoxicity of all three nps, which further justify that the linkage of treatment 2 successfully reserved the targeting effects of the anti-HER2 antibody since the targeting specificity is brought in by the HER-2 domain on cells. And the samples simply added by CH antibody showed its cell viability relevantly consistent to 1, excluding the effects of antibodies that might harm cells. However, the overall cell viability of HER2 negative cells are relevantly low, which means that there was something wrong in the control, however, this error was not necessary and could be ignored.
For treatment 2 nanoparticles, the time period was changed into 4h. For the second figure, all the
nps exhibited obvious cytotoxicity since the cell viability showed evident decline as the
concentration of nps increased, which ndicates the cytotoxicity of all three samples in T2 to be
concentration-dependent. And in the third figure of HER2 positive cells, there did showed almost a
similar trend to the 2h-nps HER2 positive cells.
For treatment 2 nanoparticles, the first figure of the BT-474_bio cells, the cell viability of all
the groups were relatively low.
And for the MDA-MB-231 in second figure, there did not exist obvious difference between bio+AB, cell control, and CH(antibody), which were relatively closed to 1.0.
While the cell viability of the group bio-unlinked was significantly lower than other groups. Compared two figures, the total average cell viability in MDA-MD-231 was relatively lower than the BT-474. And there was no obvious evidence to show the relationship.
Figure 1. cytotoxicity test for her-2 negative cell(MDA-MB-231).
Figure 2. cytotoxicity test for her-2 negative cell(BT-474).
Chem-AB represents Chemically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody with poly-his tag attached at the end.
Bio-CH represents Biologically Synthesized Nanoparticle Linked with scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.
CH represents scFv domain of anti HER2 antibody with cystine-poly-his attached at the end.
AB represents scFv domain of anti HER2 antibody with poly-his tag attached at the end.
For treatment 1 nanoparticles, the first figure of the test with the her-2 negative cells MDA-MB-231, all the groups exhibited obvious cytotoxicity for the cell viability of all groups showed low. And there did not exist considerate difference between control, bio-unlinked. Bio-AB, and bio-CH, as the cell viability were all relatively closed to 1.0.
And for her-2 positive cell BT-474 in the second figure, there did not show the obvious difference except chem-AB and chem-CH. The cell viability of adding chem-AB and chem-CH were lower than other groups significantly.
Compared two diagrams, there was no obvious change in cell viability in the two groups with the addition of chem-AB, which was convinced the cytotoxicity of the chem-AB. While the targeting effects of chem-CH were exhibited, as there did exist a obvious difference between positive cell BT-474 and negative cell MDA-MB-231 with cell viability in figure 1 was significantly higher than the figure 2.
Figure 3. Treatment1 chem-synthesis nps cytotoxicity test(HER2 positive cells).
Figure 4. Treatment1 chem-synthesis nps cytotoxicity test(HER2 negative cells).
Chem_nps_1 and chem_nps_2 represent chemically synthesized nanoparticles with different batches.
For treatment 1 nanoparticles, the first figure of the test with the her2 positive cells BT-474, all the nps exhibited obvious cytotoxicity since the cell viability showed evident decline as the concentration of nps increased, which indicates the cytotoxicity of all three samples in T1 to be concentration-dependent. These nps showed a relevantly large cytotoxicity (close to 1) at low concentrations. And there did not exist considerate differences between cytotoxicity of nps-2 linked to antibodies and bare nps-2. Also, for her2 negative cells MDA-MB-231 in the second figure, all three nps showed similar effects of lowering the cell viability with increased concentrations. These results can be roughly concluded that the nps have great killing effects on these two breast cancer cells and the linkage of antibodies by treatment 1 could have failed to equip our chemical synthesized nps with expected specificity to her2 positive cells, which means the targeting effects of the anti-her2 antibodies do not seem to function after linkage.
Figure 5. Treatment2 chem-synthesis nps cytotoxicity test(HER2 positive cells).
Figure 6. Treatment2 chem-synthesis nps cytotoxicity test(HER2 negative cells).
For treatment 2 nanoparticles, figure 1 shows the killing effects of nps on the her2 positive cells BT-474. The cell viability showed mild decline as the concentration of nps with antibodies AB and CH increased, which indicates the cytotoxicity of all three samples in T2 to be concentration-dependent as well. And the curve of cell viability of bare antibodies here does not show evident trend, differing from nps linked to antibodies. Since the cytotoxicity of chemically synthesized nps has been proved in previous experiments, this result indicates nps linked to antibodies successfully targeted HER2 positive breast cancer cells. The subsequent tests conducted on HER2 negative breat cancer cells MDA-MB-231 (in figure2) showed limited tendency of concentration-dependent cytotoxicity of all three nps, which further justify that the linkage of treatment 2 successfully reserved the targeting effects of the anti-HER2 antibody since the targeting specificity is brought in by the HER-2 domain on cells. And the samples simply added by CH antibody showed its cell viability relevantly consistent to 1, excluding the effects of antibodies that might harm cells. However, the overall cell viability of HER2 negative cells are relevantly low, which means that there was something wrong in the control, however, this error was not necessary and could be ignored.
Figure 7. BT-474 cytotoxicity test result (HER2 positive cells).
Figure 8. 4h-Treatment1 chem-synthesis nps cytotoxicity test (HER2 positive cells).
Figure 9. 4h-Treatment2 chem-synthesis nps cytotoxicity test (HER2 positive cells).
Figure 10. 2h Cytotoxicity of Biologically Synthesized Nanoparticle to BT-474
Figure 11. 2h Cytotoxicity of Biologically Synthesized Nanoparticle to MDA-MB-231
And for the MDA-MB-231 in second figure, there did not exist obvious difference between bio+AB, cell control, and CH(antibody), which were relatively closed to 1.0.
While the cell viability of the group bio-unlinked was significantly lower than other groups. Compared two figures, the total average cell viability in MDA-MD-231 was relatively lower than the BT-474. And there was no obvious evidence to show the relationship.
His-tagged protein purification [7]
Method Description
In our vector design for scFv domain of anti-HER2 antibody, we added a 6 × His-tag
to the terminal of the scFv domain of the anti-HER2 antibody.
Therefore, we use the HisTrap HP His tag protein purification column and the Union AKTA system to purify the His-tagged protein from the cell lysate.
In our case, we are trying to express a eukaryotic gene in prokaryote E. coli, therefore, we chose the SHuffle strain for better disulfide bond formation in the protein expressed to best maintain the proper protein structure and avoid the formation of inclusionbody.
After the expression of the His-tagged protein, cell pellets were broken by high pressure homogenizer and the cell lysate were collected.
Then, the cell lysate after dislodging the cell debris by centrifugation were loaded into the HisTrap HP His tag protein purification column and the His-tagged protein were eluted by imidazole.
Therefore, we use the HisTrap HP His tag protein purification column and the Union AKTA system to purify the His-tagged protein from the cell lysate.
In our case, we are trying to express a eukaryotic gene in prokaryote E. coli, therefore, we chose the SHuffle strain for better disulfide bond formation in the protein expressed to best maintain the proper protein structure and avoid the formation of inclusionbody.
After the expression of the His-tagged protein, cell pellets were broken by high pressure homogenizer and the cell lysate were collected.
Then, the cell lysate after dislodging the cell debris by centrifugation were loaded into the HisTrap HP His tag protein purification column and the His-tagged protein were eluted by imidazole.
Results / Analysis
Basically, the His-tagged protein was successfully purified from the cell lysate,
including
the scFv-polyhis, scFv-cys-polyhis and scFv-polyhis-cys.
The purity of the His-tagged protein was acceptable for further use. And the concentrations of final product were in the range of 0.1-5 mg/ml, which are also proper for later use. By our method for protein expression and purification, when results are stable, we had a yield of 5 mg/ml with total volumn 100 μL from 150 mL culture, which is a satisfying result.
The other thing we found is that, for scFv-cys-polyhis protein, the induction under 18 ℃ shows higher final concentration result than that under 37 ℃. This may be due to the fact that lower temperature would leave enough time for the protein to fold properly.
The UV spectrum during purification process, the SDS-PAGE result and the concentration by BCA test could be found in the PDF file in the supplementary data.
The purity of the His-tagged protein was acceptable for further use. And the concentrations of final product were in the range of 0.1-5 mg/ml, which are also proper for later use. By our method for protein expression and purification, when results are stable, we had a yield of 5 mg/ml with total volumn 100 μL from 150 mL culture, which is a satisfying result.
The other thing we found is that, for scFv-cys-polyhis protein, the induction under 18 ℃ shows higher final concentration result than that under 37 ℃. This may be due to the fact that lower temperature would leave enough time for the protein to fold properly.
The UV spectrum during purification process, the SDS-PAGE result and the concentration by BCA test could be found in the PDF file in the supplementary data.
Supplementary Data
The supplementary data including the UV280 absorption monitoring
information, SDS-PAGE result and BCA test result
for protein purification could be seen in the pdf file shown below:
His-tagged Protein Purification
Supplementary Data