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Applications of BBa_K1891008

α-tubulin-YCE was 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.1) for plasmid amplification.

Fig.1 Result of colony PCR
Arrows show the correct size of fusion gene fragments: α-tubulin-YCE is 1650bp.

Sequencing results further confirmed that α-tubulin-YCE expression vectors were constructed successfully.

Expression vectors were transformed into E.coli expression strain TranB(DE3), and western-blot(Fig.2) were done to test the protein from supernatant, pellet and renatured inclusion body.

Fig.2 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.

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.3) and after(Fig.4) breaking the bacteria, and Western blot(Fig.5) was also applied for the further confirmation.

Fig.3 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.4 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.5 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).

Based on the results above, we could confirm that α-tubulin-YCE was successfully expressed in rossatta cell.

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