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Gene fragments of β-tubulin was amplified via PCR and verified by electrophoresis(Fig.1). The theoretic gene size of β-tubulin is 1335bp, which matched our experimental results.

Fig.1 Electrophoresis result of β-tubulin gene fragments

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

Fig.2 Electrophoresis result of β-tubulin expression vectors
（B: electrophoresis result of colony PCR. The arrows show the correct size of β-tubulin.

Expression vectors were transformed into E.coli expression strain TranB(DE3). After culturing, we firstly tested the effect of IPTG inducement. SDS-PAGE(Fig.3) showed that IPTG is very significant in the expressing process.

Fig.3 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.

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.4) and after(Fig.5) breaking the bacteria, and Western blot(Fig.6) was also applied for the further confirmation.

Fig.5 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.6 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.7 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 β-tubulin was successfully expressed in rossatta cell.

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