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Applications of BBa_K1891007

α-tubulin-YNE 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-YNE is 1866 bp.

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

Expression vectors were transformed into E.coli expression strain TranB(DE3).We checked the protein expression predicted website http://www.biotech.ou.edu/. It showed that our fusion protein would probably expressed as inclusion bodies. We therefore renatured the inclusion bodies and verified through SDS-PAGE(Fig.2).

Fig.2 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.3) were done to test the protein from supernatant, pellet and renatured inclusion body.

Fig.3 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.4) and after(Fig.5) breaking the bacteria, and Western blot(Fig.6) was also applied for the further confirmation.

Fig.4 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.5 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.6 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-YNE was successfully expressed in rossatta cell.

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