Overview

Our project focuses on the drug screening of anti-cancer medicines, especially those that can prevent the growth of tumor cells by inhibiting the disaggregation of microtubule. The existing methods to extract microtubule are quite expensive and complex. What’s more, observing the aggregation or disaggregation level of tubulin requires electron microscope or spectrometer which can measure the light absorption in 350nm. Enormous inconveniences of using such equipment are obvious, not to mean the low accuracy in measurement. Based on current status , we hope to express human tubulin monomers in E.coli prokaryotic expression system, and use FLC (Firefly luciferase complementation) or BiFC (Bimolecular fluorescence complementation) to detect the aggregation degree of tubulin monomers in vitro. Under visible spectrum, the detection should be more easy and sensitive.

Taxol is widely used among anti-cancer medicines. It can inhibit disaggregation therefore stabilize the tubulin^[1][15], preventing the tumor cells from growing. Based on this principle, we plan to use our designed novel system to detect the existence of taxol, and hope to quantify its concentration through fluorescence intensity.

In order to achieve our goal, We ligate N-luciferase and C-luciferase (or YNE and YCE) to α-tubulin respectively for n-luc-α-tublin (YNE-α-tublin) and c-luc-α-tublin (YCE- α-tublin) vectors. We also construct β-tublin vector which can express β-tubulin monomer. All these vectors are transformed into E.coli TransB (DE3) cells for the expression of our objective proteins (Figure 0.1).

After expression and purification of α-tubulin (linked with N/C terminal of signaling proteins) and β-tubulin, we mix them in vitro and add taxol sample. Fluorescence intensity will tell the concentration of taxol or its analogues. (Figure 0.2). Meanwhile, a normalized kit will be designed as our final product.

Because the protein sequences we targeted are from human breast cell, which may have some rare codons. These rare codons may lead to the abnormal expression of tubulin in prokaryote. In order to solve this problem, we use E.coli Rossatta(DE3) as our expression strain ^[2][3].

Our PART-design can be divided into three groups

1. α-tubulin，β-tubulin expression parts.
2. FLC-based fusion protein expression parts.
3. BiFC-based fusion protein expression parts.

As a control group of our project, we extract microtubule from porcine brain to explore the conditions of tubulin aggregation in vitro, which also provide important experimental data to our modeling part.Click to see.

Project

1.Expression of α-tubulin、β-tubulin

Taxol plays an important role in mammalian tubulin aggregation, the mainly interaction sites are K19、V23、D26、H227、F270 on β-tubulin^[4]. After analyzing the sequence from human breast cancer cell, we determine that tubulin-taxol interactions exist theoretically. Thus we design and synthesize primers based on the sequence of human β-tubulin, adding Hind III and Xho I restrictive sites to the 5' and 3' flanked sites respectively. We extract mRNA from Mcf7 (human breast cancer cell line), obtain cDNAs via reverse transcription and use these as PCR templates to get a-tubulin, β-tubulin respectively.

We ligate α -tubulin, β-tubulin to pET30a(+) vector. The target genes were on the up stream of his-tag and down stream of T7 promoter, and transform the constructed vectors into E.coli TransB(DE3) to express our protein^[12][13][14]. After expression, a-tubulin and β-tubulin aggregate spontaneously in vitro^[6].

We collaborate with FAFU-CHINA. They help us to verify whether our constructed vectors can express active tubulin monomers in prokaryotic system.

Their team plan to use Co-Immunoprecipitation(CoIP) to confirm their interaction. Since pET30a(+) has His protein tag, they plan to add HA protein tag and Flag protein tag to the down stream of α-tubulin and β-tubulin respectively . The target fragments are amplified by PCR and cloned to T vector and sequenced. Then they link the confirmed gene to pET30a(+) (enzyme site: XhoI, HindIII) .After that，they transform the recombination vector into expression strains(BL21) for culturing. When the concentration of bacterium is appropriate (OD is 0.6-0.8), they induce the strains by 1 mmol IPTG. By using ultrasonic waves to break the cell of bacteria and centrifuging, they obtain the pellet and deal with the inclusion body for the further experiment of Co-Immunoprecipitation(CoIP). （see more in our collaboration.Click to see.）

2.FLC based fusion protein design

According to previous research, taxol can not only longitudinally stabilize the dimer in protofilament but also laterally stabilize neighboring protofilaments^[1][4].Since taxol interacts with β-tubulin^[4], we design to fuse luciferase fragments to α-tubulin in order to avoid the potential steric effects.

The luciferase was split into N-terminal (amino acids 1~416) and C-terminal (amino acids 417~570) fragments. When treated with luciferin B, two sides of luciferase could combine and emit fluorescence in 560nm wavelength. Thus, we design and synthesize primers based on the sequence of luciferase, using FLC plasmids pCambia1300-N-Luciferase and pCambia1300-C-Luciferase as PCR templates to get the gene sequence of n-luciferase and c-luciferase respectively.

We design primers to add EcoR I as well as Xho I restriction enzyme sites at two sides of n-luciferase and c-luciferase. We ligate n-luciferase and c-luciferase fragments to pET30a(+) vector. The target genes are on the up stream of his-tag and downstream of T7 promoter. After constructing the vectors successfully, we transform them into E.coli TransB(DE3) for protein expression. We use Ni beads to purify the expressed proteins. Purified n-luciferase and c-luciferase are mixed together, after that, we add luciferin B into the system and test the light absorption at 560nm via spectrophotometer. The intensity of background light when monomers do physical collision in solution can be calculated, and we can verify whether active n-luciferase and c-luciferase can be expressed in prokaryotic expression system.

Fusion PCR technology is applied in the further construction of our bio-bricks. The n-luciferase is ligated to N/C terminal of α-tubulin respectively. Similarly, c-luciferase is ligated to N/C terminal of α-tubulin. These four sequences above are the foundation of expressing “α-tubulin-nluc” fusion protein and “α-tubulin-cluc” fusion protein. Moreover, a GGGGSGGGGSGGGS protein linker is also established to the fragment between two target genes. （Figure 2.1）Then, we ligate these fragment to pET30a(+) vector, expressing and purifying our target proteins.

At the same time, we use Gateway large-scale cloning technology to construct nluc-β-tubulin and β-tubulin-cluc gene sequences, ligating them to pET30a(+) expression plasmid.（as shown in Figure 2.1） Our target genes are inserted between T7 promotor and his-tag. Nluc-β-tubulin and β-tubulin-cluc fusion proteins are expressed and purified with the same method mentioned above. Since α-tubulin and β-tubulin can combine as heterodimer spontaneously in vitro, we mix α-tubulin-cluc or cluc-α-tubulin with nluc-β-tubulin as system1, mix α-tubulin-nluc or nluc-α-tubulin with β-tubulin-cluc as system2, and then add luciferin B to both systems. Microplate reader is used to detect the absorption wavelength at 560 nm. It can be determined whether n-luciferase and c-luciferase maintain the normal function after fusing with tubulin monomer by observing the fluorescence exists or not.

The proteins who are verified successfully are applied to the further research. In order to reduce the nonsense collision between protein molecules and reduce the background fluorescence, we use semi-solid mixed crowding as the buffer^[5]. Mixed crowing can modify the inner cellular circumstance. First, we mix α-tubulin-nluc, α-tubulin-cluc with β-tubulin to get nluc-tubulin dimer and cluc-tubulin dimer respectively(Figure 2.2). Since a certain length of microtubule is needed as a premise for taxol’s function, GTP is added into the system to make the heterodimers aggregate into a loci. The light intensities of possibly combined luciferase fragments are defined as the zero point when adding luciferin B as a substrate.

We set a serial dilution of taxol as experimental groups. Taxol is mixed with tubulin heterodimer mixture and wait for a certain time, then luciferin B was added. Since the more tubulin heterodimers taking part in the aggregation activity, the more complete luciferase can exist. There would be a relationship between the intension of fluorescence and the length of microtubule（Figure 2.3）. Through modeling, we know that the length of microtubule and the concentration of taxol is positively correlated^[7], so we can fit a standard curve according to the concentration of taxol and the intension of fluorescence detected in the experiment.

3.BiFC based fusion protein design

Besides the methods mentioned above, we also use yellow fluorescence protein(YFP) to construct a similar system based on BiFC theory.

We split YFP into N-terminal (YNE, amino acids 1~155) and C-terminal (YCE, amino acids 156~240). When the two parts are expressed separately and mixed together in vivo or in vitro, they cannot aggregate into a complete protein, so no fluorescence can be detected^{[8][9][10][11]}. Once the two parts are fusing expressed with a pair of proteins who can interact with each other, YFP can regain its function. The excitation wavelength is 514 nm, emission wavelength is 527 nm. We design the N-terminal and C-terminal primers based on YFP’s sequence, using pSPYNE and pSPYCE as templates, running PCR to get YNE and YCE gene sequences.

Then we use the same design as luciferase, both of which serve as the signal proteins (Figure 3). Expression and function verification experiments are carried out then. We used spectrophotometer to detect the intension of emission fluorescence of microtubule under different taxol treatment condition with excitation wavelength of 527 nm. The standard curve of taxol concentration and fluorescence intension can be measured later.

4.Kit

Based on the design mentioned above, the final product can be carried out as a standardized kit（Figure 4）. Our kit contains 5 parts:

1. The purified α-tubulin-nluc fusion protein, α-tubulin-cluc fusion protein, α-tubulin-YNE fusion protein, α-tubulin-YCE fusion protein and β-tubulin dissolved in mixed crowding respectively.
2. GTP solution with a certain concentration.
3. Luciferin B
4. User guide
5. A standard curve for taxol concentration – intensity of excitation fluorescence.

The kit has various functions:

1. Quantitative detection of Taxol’s concentration in taxol fermentation broth.
2. Screen of anti-microtubule-disaggregation medicine.

5.Extracting tubulin in vitro

Tubulins of animal origin has the ability of self-assembly under proper condition in vitro. When GTP and Mg2+ are added into near-neutral pH buffer PIPES, tubulin heterodimers can aggregate into microtubule automatically under room-temperature (or 37℃). This process can be reversed under lower temperature (usually 4℃ in experimental condition). It is the basis to extract and purify microtubule in vitro that tubulins can assemble or disaggregate automatically under different temperatures.

As a major member of cytoskeleton, microtubule plays an extremely important role in every organism. Researches focusing on microtubule are of great significance. Porcine brains contain abundant microtubule. As an inexpensive raw material which can be easily purchased, they are always used for microtubule extraction^[16] Since microtubule is conservative in both animal and plant, the microtubules purified from animal tissues can also be used in plant science research.

To test the aggregation of microtubule effectively, we design the fusion proteins of tubulin subunit and fragments of signal proteins. However, whether the fusion proteins can maintain the same functions of the origin proteins is largely unknown. So we extract and purify the porcine origin tubulin, design a series of aggregation experiment in vitro, expecting to provide a positive control for the fusion proteins expressed from prokaryotic cells.