Part:BBa_K2306006
Cytosolic-abundant heat soluble protein 94205 (CAHS 94205)
This Biobrick features the gene sequence that encodes for the production of the tardigrade intrinsically disordered protein (TDP) "CAHS 94205". This is one of the several heat soluble proteins found in the tardigrades responsible for the ability of these microscopic animals to survive the harshest conditions. Upon desiccation, tardigrade proteins form a glass matrix capable of protecting other fragile biological components. Therefore, this tardigrade protein can be combined with other perishable biological material (e.g. proteins) to increase its stability against dehydration or even enable the possibility of stabilizing material with just a previous drying step. This BioBrick is featured in the composite part BBa_K2306011 combined with a T7 promoter BBa_R0010, a ribosome binding site BBa_k2306014 and a double terminator BBa_B0015 to control its expression. Further information about our project can be found on our [http://2017.igem.org/Team:TUDelft/Results#TDP results page].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 459
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 178
- 1000COMPATIBLE WITH RFC[1000]
Pilot Expression
In order to attain this protein, CAHS 94205 was expressed in the pET-28b(+) plasmid. We used the native sequence for CAHS 94205 for our expression and characterization. The part info is BBa_K3008003. Since the T7 polymerase is not naturally occurring in E.coli, we used BL21 Xjb(DE3) Autolysis E.coli cells (Zymo Research, n.d.). This is a strain of E.coli that produces the RNA polymerase necessary for transcription of our genes of interest when induced by the addition of IPTG.
We determined the optimal time for induction by creating a growth curve of BL21 XJb(DE3) cells containing the CAHS 94205 plasmid (Figure 1). The curve depicts the lag-phase, log-phase, and stationary phase of our cells. Figure 1 shows that the mid-log phase of our cells is at an OD600 of about 0.5 and it occurs after about 3.5hr of incubation at 37°C and shaking at 250 RPM in liquid LB medium supplemented with 50 μg/mL of kanamycin. This was conducted at the regular atmospheric pO2 of ~160 mmHg. We observed a dip in cell concentration at the 6hr time point, which may have been caused by insufficient blanking or by cell death from a lack of oxygen. When we collected samples for the 7-hour time point we noticed the liquid cultures were capped too tightly after the 6-hour samples were collected.
The growth curve indicated that we should induce our liquid cultures with IPTG after 3.5 hr of incubation or at an OD600 ranging from 0.4 to 0.6. To characterize the heat solubility of CAHS 94205 and the localization of the protein after expression, we induced cells in 10 mL LB/KAN media with a final concentration of 1mM IPTG. We collected samples hourly for six hours and analyzed the heat-soluble and insoluble fractions of the supernatant and the lysate (Figure 2). The results from SDS-PAGE indicated that CAHS 94205 was heat-soluble. From this experiment, we also determined that the 3hr, 4hr, and 6hr samples had clear 26 kDa bands compared to the other samples. The 5hr samples had suspiciously similar results to the non-induced samples. To be safe, we decided to end expression for our large scale protein production at 4hr. There are proteins left in the insoluble fractions of lysate which could have resulted from insufficient cell lysis. It is also possible that the 95°C purification step caused a portion of the produced IDPs to become insoluble.
Large Scale Production
Since the characterization of CAHS 94205 protein expression revealed a consistent success of purification in the heat-soluble lysate after 4hr of IPTG induction, we proceeded to large scale production. For large scale production, we induced 1L cultures of CAHS 94205 transformed BL21 derived E. coli cells with a final concentration of 1mM IPTG for 4hrs. The cell pellet was purified by heat solubility and a sample from the soluble fraction was tested by SDS-PAGE to verify the purity of CAHS 94205 (Figure 3). The SDS-PAGE results showed a large 26 kDa band along with a less concentrated 15 kDa band. The 15 kDa band was not present in the non-induced control, so it may be that CAHS 94205 is being cleaved in half by a protease and staying heat soluble or CAHS 94205 may be interacting with a small protein and staying heat soluble.
Purification of Large Scale
During the first phase of our project, we performed a large scale protein production and purification of native CAHS 94205. We also conducted large scale production of CAHS2 (D3), CAHS1 (D4), SAHS10 (G3), LEA1(A4), and rvLEAM (H4); all five of which contained an N-terminal FLAG-tag. After we received a product, we autoclaved them for 20 minutes to purify the proteins. The SDS-PAGE gel of the purified products shows that only CAHS D (CAHS 94205), CAHS 1 (CAHS 107838), SAHS 10 and LEA1 had good yield and withstood being exposed to a temperature of 121°C. As a result, we decided to continue site-directed mutagenesis with these four proteins.
Circular Dichroism
These data showcase a stepwise increment of temperature and the corresponding CD spectra for CAHS 94205. The data in Figure 1 suggest that as the temperature increases, the protein is becoming more disordered. The ramp down temperatures closely fall in line with their ramp up counter parts which is consistent with the lack of denaturing/agglomeration characteristics of this protein.
Further characterization on this will be done by using a less error prone solvent for the proteins, specifically a sodium phosphate buffer. Additionally, smaller temperature increments will be used to detect the inflection at which protein begins become disordered.
Red: 25C Black: 40C Green: 60C Purple: 80C Blue: 60C Down Orange: 25C Down
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