Results
Do One Thing1. The expression of α-tubulin, β-tubulin, n-luciferase, c-luciferase
1.1 Vector construction
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.
Gene fragments were ligated to E.coli expression plasmid pET30a(+), after transformation, colony PCR was done to verify the efficiency (Fig.2A and Fig.2B). Meanwhile, the sequencing results further confirmed that we successfully constructed the 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.
Expression vectors were transformed to E.coli expression strain TransB(DE3). After culturing and inducing with IPTG, bacteria were lysed and SDS-PAGE(Fig.3)/ western-blot (Fig.4) were done to test the protein from supernatant, pellet and renatured inclusion body.
(left to right: non-induced group, expressed empty vector, induced group) arrow shows the correct molecular weight of target protein(55 kDa).
(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.
Apart from this, we also transformed plasmids to Rossatta(DE3) which can express rare codons and improve the expression level of eukaryotic protein.
Before breaking the bacteria via ultrasonic waves, SDS-PAGE (Fig.5) was established to verify the results.
(left to right: protein marker, expressed empty vector, α-tubulin(55 kDa), β-tubulin(55 kDa), α-tubulin-YNE(75kDa), YNE-α-tubulin(75 kDa) ) arrows show the correct band.
After breaking the bacteria, SDS-PAGE (Fig.6) was also established to verify the results.
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).
Western blot(Fig.7) was also applied for the further confirmation.
A: left to right, protein marker, negative control, α-tubulin(55 kDa), α-tubulin-YNE(75kDa), α-tubulin-YCE(66 kDa), YCE-α-tubulin(66 kDa), cluc-α-tubulin(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).
According to Fig.7B, the target protein can be tested out in the supernatant, indicating that they are soluble when expressed in rossatta strain.
Based on the results above, we can confirm that α-tubulin and β-tubulin were successfully expressed in cell.
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.
（这里放发福的结果）
2. The expression of fusion protein
2.1 fusion PCR
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, cluc-α-tubulin were constructed respectively via fusion PCR. After ligating these fusion gene fragments to pET30a(+) with restriction enzyme, we transformed the target plasmids to Trans5α. When colony PCR was done, we picked correct colony shown in electrophoresis(Fig.8) for plasmid amplification.
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 showed that α-tubulin-YNE, YNE-α-tubulin, α-tubulin-YCE, YCE-α-tubulin, β-tubulin-YCE, YCE-β-tubulin, α-tubulin-nluc, cluc-α-tubulin expression vectors were constructed successfully.
We transformed the expression plasmids to E.coli expression strain TranB(DE3). The protein expression predicted website http://www.biotech.ou.edu/ showed that our fusion protein would probably expressed as inclusion bodies. We therefore renatured the inclusion bodies and verified through SDS-PAGE(Fig.9).
the molecular weight of target fusion protein is 74.6kDa. arrows show the correct bands.
Also, western-blot (Fig.4) were done to test the protein from supernatant, pellet and renatured inclusion body.
Apart from these, we also expressed our target protein through Rossatta(DE3) strain and used SDS-PAGE(Fig.10) to verify the expression.
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).
SDS-PAGE were done to verify the expression results before(Fig.5) and after(Fig.6) breaking the bacteria, and Western blot(Fig.7) was also applied for the further confirmation.
Based on the results above, we can confirm that 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 cell.
2.2 Gateway
In our experiment, we also try to construct fusion protein vectors with Gateway Large-scale Cloning technology. We used Invitrogen pENTR/D TOPO to clone β-tubulin into entry vector. Designing primer based on β-tubulin sequence and running PCR procedure. From this picture, the band of β-tubulin was correct.
In order to do LR reaction, we used the restriction endonuclease not I to digest the entry vector. The bands of linear vector and the origin vector suggested that the digestion was efficiency.
Using Invitrogen Gateway LR Clonase II Enzyme Mix, the entry vector can be ligate with pCambia1300-nluc and pCambia1300-cluc respectively. So the destination vectors were complete. After transformation, running PCR with β-tubulin’s primers, the bands show high positive rates as showed in Fig.13
Extracting plasmid, the product gal bands show that cluc-β-tubulin is correct. Running PCR using the reverse primer of β-tubulin and the forward primer of cluc, the correct band existed.
arrows show the correct bands
3. Results of tublin extraction in vitro
After successfully extracting tubulin from porcine brains, we tried to summarize the aggregation conditionin vitro by using electron microscope.
From pictures taken under the electron microscope(Fig.15), we could see tubulin (treated with 1μM 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 1 OD350 of microtubule samples treated with serial concentration of taxol
| Taxol concentraion(μM) | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| 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 1, 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.
