Expressing

and Characterizing the Target of Rapamycin-Kinase Domain of

Chlamydomonas reinhardtii in Escherichia coli
Amita Bollapragada

Overview Methods Conclusions

Algae are a promising feedstock for biofuels due to their lipid content, Using SDS-PAGE and LC-MS/MS we were able to verify the
which is a large component of biofuels.1 Kinases control a large number 2. 3. 5. presence of TOR KD in our samples, and consequently
of essential biological pathways in many organisms, including the 1. 4. confirm that our recombinant method of protein expression
network that produces lipids in algae.1 Being able to individually and purification were successful. We were then able to use
analyze how a specific kinase contributes to the kinase network that the Universal Kinase Assay Kit to determine that our TOR KD
produces lipids will give us a better understanding of this network. This kinase sample was active. Now that we have this information,
can possibly help to produce larger amounts of lipids to sustain the we will be able to further analyze the kinase to determine the
growing fuel demands of the world. substrates it reacts with, and its function in the kinase
To be able to individually analyze our kinase of interest, we used a network of Chlamydomonas reinhardtii. By doing this, we can
recombinant method of protein expression to obtain a purified active
9. 8. 7. 6. develop our understanding of this network that produces
sample of our kinase. A cell lysate was prepared and purified from lipids in this green algae. This can possibly help to produce
Escherichia coli cells that have been induced to express the Target of larger amounts of lipid, a large component of biofuels, to
Rapamycin kinase domain (TOR KD), originally from the green algae sustain the growing fuel demands of the world.
Chlamydomonas reinhardtii. We then separated and visualized our
purified proteins using Sodium dodecyl sulfate-Polyacrylamide Gel
Electrophoresis (SDS- PAGE) and liquid chromatography- tandem mass
References
spectrometry (LC-MS/MS) to confirm the presence of our kinase. We 1. Plasmids containing the TOR-KD coding sequence and glutathione S-transferase (GST) tags were transformed into a p-GEX6P-1 vector and inserted into a 1.Jeong-jin Park; Wang, H.; Gargouri, M.; Deshpande, R. R.; Skepper, J. N.;
were able to use these methods to verify the presence of TOR KD in our BL21(DE3) expression strain of E. coli. Cultures were then grown in ampicillin-containing LB broth and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to Holguin, O.; Juergens, M. T.; Hill, Y. S.; Hicks, L. M.; Gang, D. R.; The Response of
samples, and consequently confirm that our recombinant method of express TOR-KD. 2. The E. coli cells were mechanically lysed by a freeze-thaw cycle and chemically lysed with lysozyme. The GST-tagged proteins were then Chlamydomonas reinhardtii to Nitrogen Deprivation: A Systems Biology
Analysis. The Plant Journal, 2015, 81(4), 611–624.
protein expression and purification were successful. Finally, we used separated from the cell lysate by affinity purification with reduced glutathione. 3. The Bradford assay was performed to create a calibration curve, which was used 2.Bradford M. M., Wang R. A rapid and sensitive method got the quantification
the Universal Kinase Assay Kit to check whether our kinase has been to quantify the amount of TOR-KD. 2 4. SDS-PAGE was performed using a 4-20% gradient gel in order to separate TOR-KD from the other proteins. TOR-KD was of microgram quantities of protein utilizing the principle of protein-dye
expressed in its active form, and verified that it has. Now we can identified by comparing its distance traveled to that of a marker with known molecular weight. 3 5. The TOR-KD proteins were reduced with dithiothreitol (DTT) binding. Analytical Biochemistry, 2007, 72(1), 248-254.
further analyze the kinase to determine the substrates it reacts with, and alkylated with iodoacetamide (IAM) to prevent disulfide bonding. Trypsin was then used to digest the proteins into tryptic peptides at their R and K residues. 3.Muyzer G.; Waal C.; Ulitterlinden G.; Profiling of Complex Microbial
Populations by Gradient Gel Electrophoresis Analysis. Applied and
and its function in the kinase network of C. reinhardtii, to develop our 6. The peptides were then extracted and desalted in ZipTip format. 7. The peptides then underwent LC-MS/MS analysis, using a Waters NanoAcquity UPLC system Environmental Microbiology, 2003, 59 (3), 695-700.
understanding of this network that produces lipids. coupled to an AB Sciex TripleTOF 5600 mass spectrometer. 8. A database search using Mascot was performed to identify the proteins from the results of the LC- 4.Srinivasan J.; Cload S.; Kurz J.; ADP-Specific Sensors Enable Universal Assay of
MS/MS analysis. 9. An R&D Universal Kinase Activity Assay kit was used on a separate protein elution sample to determine the activity of TOR-KD.4 Protein Kinase Activity. Chemistry and Biology, 2004, 11(4), 499-508.

Results and Discussion

Figure 4 shows the MS1 and MS2 spectra for TOR-KD. The peaks Figure 7. Corrected
The Bradford assay uses the shift of maximum absorbance, from 465 nm to 595
of these spectra correspond to when the most abundant protein average absorbance at
nm, in a solution of Coomassie Brilliant Blue G-250 dye as the dye changes from
species were eluted during LC, and those of which were identified 620 nm (subtracting
its red, unbound form to its blue protein-bound form.2 The calibration curve
are listed (Figure 5). TOR-KD (Cre2000_pac.3.g...) was the fourth blank values) of a TOR
(Figure 1) was generated from a series of standards with a known protein
most abundant protein found, which provides good evidence that KD kinase assay with a
concentration. Using this equation and the absorbance of our sample (0.454),
the excised band from SDS-PAGE contained TOR-KD. Other 50 mg library
the protein concentration was found to be 7.42 x 10-3 mg/mL.
abundant proteins included naturally occurring proteins found in of extracted
A graph of distance traveled versus log(MW) was E. coli, which were expected to be detected. However, the most Chlamydomonas
generated for each band in the ladder (Figure 3). The Figure 4. Total Ion Chromatogram for abundant proteins found were contaminants, mostly consisting of peptides.
. Figure 1. Bradford Assay Calibration Plot equation of this plot was found to be y=-3.97x + 11.1 the protein elution sample. human keratin (skin) proteins, such as the most abundant We were able to use mass spectrometry and SDS- PAGE to verify
of BSA standards at an absorbance of 595 nm. (where ‘y’ is distance traveled (cm)] and ‘x’ is protein K2C1_HUMAN. These contaminants were most likely the presence of TOR KD in our samples. However, in order to
log(MW)). Using this equation, the MW of the introduced during the SDS-PAGE as a result of the experimenters’ actually study the function of TOR KD, we had to confirm that it
proteins corresponding to the darkest bands in the skin cells coming into contact with the gel. There was also a sheep had been expressed in its active form. The Universal Kinase Assay
post-induction, and total protein lane, was computed keratin contaminant (K1C15_SHEEP), which most likely came from Kit (R&D Systems) was used to measure ADP formation. This kit
to be 61.3 kDa. the fibers of the experimenters’ lab coats. The presence of these provides a simple, compatible method for assaying the activity of
This value was then compared to the theoretical proteins is a source of experimental error because they input kinases in vitro. if the kinase is active then it will phosphorylate
mass of TOR KD (56 kDa), which is relatively Figure 5. Ten most abundant more contaminant ions into the MS, which makes it more difficult specific substrates to form ADP, which we will correlate to kinase
close. However, because there was an absence proteins in the elution sample. to detect the ions of interest. This results in fewer peptide activity using the Universal Kinase Assay kit through the use of
of the bands corresponding to our protein in the matches and thus less protein sequence coverage, meaning less malachite green reagents. The more activity a sample has, the
soluble protein, and elution lane, the bands accurate results. The protein sequence coverage was 25% and had darker the green and the higher the absorbance value.4 The TOR
Figure 2. SDS-PAGE gel images observed in the post-induction and total protein a relatively even distribution of matched peptides throughout the KD assay had an absorbance of 2.40 for the positive control and
Figure 3. Plot showing the log of the using Coomassie staining, showing lane could actually be false positives from sequence (Figure 6). Despite the presence of contaminants, this 1.80 for the kinase sample. This value for the kinase sample is far
molecular weight versus the distance Figure 6. Protein sequence coverage amount of coverage provides good evidence that TOR-KD is
molecular weight markers (ladder) proteins that are the same MW as TOR KD. To above the value for the negative control (0.44) (Figure 7). Hence,
traveled (mm) of the molecular weight and protein samples. verify the presence of our protein, we used for the elution sample. present and has been correctly identified. we concluded that our TOR KD kinase sample was active.
markers through the SDS-PAGE gel. another analytical method, mass spectrometry.

Department of Chemistry, University of North Carolina, Chapel Hill, NC USA