Vector Construction

Vectors of α-tubulin, β-tubulin, n-luciferase, c-luciferase

Gene fragments of α-tubulin, β-tubulin, n-luciferase, c-luciferase were amplified via PCR and verified by electrophoresis(Fig.1). The theoretic gene size of α-tubulin is 1356bp, β-tubulin is 1335bp, n-luciferase is 1248bp, c-luciferase is 459bp, which matched our experimental results.

Fig.1 Electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase gene fragments

Gene fragments were ligated to E.coli expression plasmid pET30a(+), after transformation, colony PCR was done to verify the efficiency(Fig.2A and 2B). Meanwhile, the sequencing results further confirmed that we successfully cloned the α-tubulin, β-tubulin, n-luciferase, c-luciferase expression vectors.

Fig.2 Electrophoresis result of α-tubulin, β-tubulin, n-luciferase, c-luciferase expression vectors
(A: electrophoresis result of colony PCR. The arrows show the correct sizes of α-tubulin, n-luciferase and c-luciferase.
B: electrophoresis result of colony PCR. The arrows show the correct size of β-tubulin.

Fusion Protein Vectors

By fusion PCR technology

α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YNE, YNE-β-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, nluc-α-tubulin, α-tubulin-cluc and cluc-α-tubulin were cloned respectively via fusion PCR. After ligating these fusion gene fragments to pET30a(+) empty vectors, we transformed the target plasmids to Trans5α. When colony PCR was done for screening, we picked correct colonies shown in electrophoresis gel(Fig.3) for plasmid amplification.

Fig.3 Result of colony PCR
Arrows show the correct size of fusion gene fragments: α-tubulin-YNE is 1866 bp, α-tubulin-YCE is 1650bp, β-tubulin-YCE is 1629bp, α-tubulin-nluc is 2640bp, α-tubulin-cluc is 1857bp.

Sequencing results further confirmed that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc and cluc-α-tubulin expression vectors were constructed successfully.

By Gateway Technology

We also tried to construct fusion protein vectors by Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Primers were designed based on β-tubulin sequence and PCR was done for verification. Electrophoresis result (Fig.4) showed that β-tubulin was successfully cloned into the entry vector.

Fig.4 PCR verification result of the constructed entry vectors

In order to do LR reaction, we used the restriction endonuclease Not I to digest the entry vector. Electrophoresis result (Fig.5) showed that single digestion was efficient.

Fig.5 Single endonuclease digestion result of entry vectors

Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vectors were ligated with pCambia1300-nluc and pCambia1300-cluc respectively. Thus the destination vectors were complete. After transformation and running PCR with β-tubulins primers, electrophoresis result (Fig.6) showed high positive rates, indicating β-tubulins was successfully cloned into the vectors.

Fig.6 PCR verification result of the objective vectors

Also, signaling fragments were also needed to be tested. By using the reverse primer of β-tubulin and the forward primer of cluc for PCR verification, we found that cluc-β-tubulin fusion protein vector is successfully constructed. Electrophoresis result is shown in Fig.7.

Fig.7 PCR verification result of pCambia-nluc, pCambia-cluc
Arrows show the correct bands

In conclusion, we successfully cloned nine fusion protein vectors. α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, and cluc-α-tubulin were ligated to pET30(+) . β-tubulin was cloned to pCambia-cluc plasmid as a form of cluc-β-tubulin fusion protein vector.

Protein Expression

In TransB(DE3) E.coli expression strain

Expression vectors were transformed into E.coli expression strain TransB(DE3). After culturing, we firstly tested the effect of IPTG inducement. β-tubulin was taken as an example. SDS-PAGE(Fig.8) showed that IPTG is very significant in the expressing process.

Fig.8 SDS-PAGE result of β-tubulin inducement test (left to right: non-induced group, induced group), arrow shows the correct molecular weight of target protein(55 kDa)
the molecular weight of target fusion protein is 74.6kDa. arrows show the correct bands.

Then we checked the protein expression predicted website It showed that our fusion protein would probably expressed as inclusion bodies. We therefore renatured the inclusion bodies and verified through SDS-PAGE(Fig.9).

Fig.9 SDS-PAGE of renatured inclusion bodies from α-tubulin-YNE, YNE-α-tubulin
the molecular weight of target fusion protein is 74.6kDa. arrows show the correct bands.

Also, western-blot(Fig.10) were done to test the protein from supernatant, pellet and renatured inclusion body.

Fig.10 Western blot result of prokaryotic expression
Left to right, extracted α-tubulin, expressed empty vector, α-tubulin,α-tubulin-YNE fusion protein, α-tubulin-YCE fusion protein, α-tubulin-nluc fusion protein. Arrows show the correct bands of target proteins, triangles show the homologous tubulin protein(FtsZ,43kDa) from the bacteria.

In Rossatta(DE3) E.coli expression strain

Rossatta(DE3) is a kind of E.coli strain that can express rare codons and improve the expression level of eukaryotic protein. Thus we applied this strain to optimize our protein expression.

SDS-PAGE were done to verify the expression results before(Fig.11) and after(Fig.12) breaking the bacteria, and Western blot(Fig.13) was also applied for the further confirmation.

Fig.11 SDS-PAGE of centrifuged cells before ultrasonic breaking.
A: cluc-α-tubulin(74 kDa), α-tubulin-nluc, YCE-β-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), α-tubulin-YCE(66 kDa), expressed empty vector.
B: left to right: expressed empty vector, α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa).
Arrows show the correct bands.
Fig.12 SDS-PAGE of supernatant after ultrasonic breaking the rossatta cells
Left to right: expressed empty vector, α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75kDa), α-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), β-tubulin-YCE(66 kDa), YCE-β-tubulin(66 kDa), α-tubulin-nluc, cluc-α-tubulin(74 kDa)
Fig.13 Western blot of rossatta cells expression.
A: left to right, protein marker, negative control, α-tubulin in pellet(55 kDa), α-tubulin-YNE in pellet(75kDa), α-tubulin-YCE in pellet (66 kDa), YCE-α-tubulin in pellet (66 kDa), cluc-α-tubulin in pellet (74 kDa), extracted α-tubulin(55 kDa). B: left to right: negative control in pellet, β-tubulin in pellet(55 kDa), β-tubulin-YCE in pellet (66 kDa), protein marker, negative control in supernatant, β-tubulin in supernatant (55 kDa), β-tubulin-YCE in supernatant (66 kDa), extracted β-tubulin(55 kDa).

Based on the results above, we could confirm that α-tubulin, β-tubulin, α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, cluc-α-tubulin fusion protein were successfully expressed in rossatta cell.

Particularly, according to figure 7B, the target proteins (β-tubulin and β-tubulin-YCE) can be tested out in the supernatant, indicating that they are soluble when expressed in rossatta strain.

We collaborated with Fujian Agriculture and Forest University and asked them to test the interaction between α and β-tubulin. Thus verified the activity of tubulin monomers.

Fluorescence detection

Protein functional test

In order to make sure the protein expressed from prokaryotic cells had their functions. We did a functional test at first. Concentrated supernatant of α-tubulin-YNE and α-tubulin-YCE were mixed equally and treated with twice as much as the supernatant of β-tubulin. 50μM GTP and 200μM taxol were also added for helping aggregation. After incubated in 37℃ for 1 hour,the mixture was tested by the absolute recording spectrofluorometer. The result(Fig.14) showed there was a significant emission peak in 525nm with the excitation in near 512nm wave length, indicating that YCE and YNE protein fragments were combined as a whole YFP and thus verifying their biological functions in vitro.

Also, equally mixed α-tubulin-YNE and β-tubulin-YCE treated with 50μM GTP and 200μM taxol indicated the existed α-β tubulin interaction and the YNE-YCE combination.

Fig.14 Fluorescence intensity - Wave length diagram

Taxol-concentration based assay

After protein functions were confirmed, we further tested the microtubule aggregation level by treating serial concentration of taxol samples. Since the excess amount of substrate proteins (α-tubulin-YNE, α-tubulin-YCE and β-tubulin) were treated. The aggregation level could be represented by the fluorescence intensity. The experimental results (Fig.15 Table 1) showed that there is an obvious positive correlation between taxol concentration and fluorescence intensity when the substrate proteins are abundant.

Fig.15 Fluorescence intensity - Wave length diagram
Table 1 Statistics of serial taxol concentration-integration of fluorescence intensity
Taxol concentration(μM) Fluorescence intensity(CPS)
0 31880000
0.5 33211000
5 33951000
25 34267000
50 35650000
100 35861000
250 42479000
500 51428000

Further visualized experiment was also carried out. As the aggregated tubulins could be centrifuged in room temperature while the tubulin monomers could not. As enough protein substrates (α-tubulin-YNE, α-tubulin-YCE and β-tubulin) existed, we centrifuged each experimental group. Results(Fig.16) showed obvious difference between each group with serial taxol concentration, further indicating that the taxol concentration can be represented by the tubulin aggregation level.

Fig.16 Pellet from each experimental group with serial taxol concentration

Based on the statistics above, any random concentration of taxol could be figured out in the following Polynomial interpolation curve(Fig.17).

Fig.17 Fluoroscence intensity - Taxol concentration polynomial interpolation curve

Results of tublin extraction in vitro

After successfully extracting tubulin from porcine brains, we tried to summarize the aggregation condition in vitro by using electron microscope.

Fig.18 Aggregated microtubule treated with 1μM taxol

From pictures taken under the electron microscope(Fig.18), we could see tublin (treated with 1 μM taxol) in aggregated form obviously, indicating we have achieved the aggregation process in vitro. However, due to the high concentration of our extracted sample, it was hard to tell the aggregated length and the quantity of microtubules. Thus we tried to use spectrophotometer to measure OD350 of our experimental samples.

Table 2 OD350 of microtubule samples treated with serial concentration of taxol

OD350 Experiment Group
1 2 3 4 5
Taxol concentration(μM)
0 0.095 0.077 0.025 0.104
0.001 0.062 0.123 0.119 0.086 0.149
0.01 0.152 0.138 0.129 0.060 0.081
0.1 0.096 0.106 0.123 0.082 0.134
1 0.148 0.140 0.149 0.061 0.092
2 0.047 0.093 0.108 0.052 0.080
3 0.068 0.091
4 0.035 0.050
5 0.020 0.059 0.140 0.078 0.100
6 0.079 0.112
7 0.076 0.076
8 0.050 0.067
9 0.079 0.107
10 0.053 0.111 0.185 0.086 0.100
12.5 0.077 0.065
20 0.028 0.099 0.108
25 0.076 0.097
30 0.093 0.164 0.154
50 0.043 0.162 0.096 0.074 0.090
70 0.113 0.188 0.156
100 0.072 0.088

From the results shown in table 2, we found that there was no obvious relationship between OD statistics and taxol concentration. The reason may be the machine issue. Due to the wave length for measuring OD is 350nm, which is between the ultraviolet light and visible light, there is a high requirement for instruments and always leads to a huge deviation. As the high technologic instruments could not be owned by every laboratory in different areas, our fusion proteins which can detect the relatively accurate concentration of anti-microtubule drugs will have a broad application prospect.