Envelope Protein (E protein)
A plasmid containing the whole structural protein of the dengue virus was obtained from National Health Research Institute. The Envelope protein (E protein) with two HA tags from the plasmid were amplified using PCR. Figure 6. shows that the sequence of the E protein with the HA tags were amplified, matching the expected size of 1,551 bp.
Figure 6. E protein with two HA tags, matching the expected size of 1,551 bp. M = DNA marker.
We prepared pET-29a(+) as the vector for expressing E protein and confirmed in Figure 7. that plasmid extraction shows the size of pET-29a(+) is about 5,000 bp, which is close to the theoretical size, 5,371 bp. We attempted to transform the pET-29a(+)_E protein into DH5α and check the transformation using colony PCR. However, Figure 8. shows the results not what was expected, and the size of the plasmid was wrong, so the ligation and transformation failed. We are trying to fix this.
Figure 7. Plasmid extraction of pET-29a(+), matching the expected size of about 5,000 bp. M = DNA marker.
Figure 8. Colony PCR of pET-29a(+)_E protein, which only has a size of about 300 bp rather than the expected 500 bp. M = DNA marker. P = positive control, pET-29b(+)_CLEC5A.
CLEC5A
A plasmid containing the CLEC5A extracellular domain with a Myc tag was obtained from OriGene Technologies. The CLEC5A extracellular domain and Myc tag were amplified using PCR. Figure 9. shows that the sequence of the CLEC5A extracellular domain with a Myc tag were amplified successfully, with a size of 564 bp. pET-29b(+) was prepared successfully as a vector, as shown in Figure 10., with a theoretical size of 5,370 bp. pET-29b(+)_CLEC5A was obtained by inserting the CLEC5A into pET-29b(+) and then transforming it into DH5which was confirmed using colony PCR. Figure 11. shows the size of CLEC5A extracellular domain with Myc tag, and T7 promoter and terminator is about 900 bp, close to the theoretical size of 904 bp.
Figure 9. CLEC5A with a Myc tag, matching the expected size of about 600 bp. M = DNA marker.
Figure 10. Plasmid extraction of pET-29b(+), matching the expected size of about 5,000 bp. M = DNA marker.
Figure 11. Colony PCR of pET-29b(+)_CLEC5A, which has an expected size of about 900 bp including the CLEC5A extracellular domain and Myc tag and T7 promoter and terminator. M = DNA marker.
pET-29b(+)_CLEC5A was then transformed to BL21(DE3) and induced to express protein with 1.8 ml bacteria and 1 M IPTG for 2 hours. Figure 12. shows the results from four different colonies with and without induction. The strong bands from 28 to 35 kDa indicate expression of the CLEC5A extracellular domain with the Myc tag, which has a size of 33 kDa. The results were also confirmed using Western blot based on the HA tag, which is part of pET29b(+). Figure 13. shows that the anti-His tag binds to the His tag, suggesting that the protein expression was successful.
Figure 12. SDS-PAGE of CLEC5A from a small-scale culture. After induction with IPTG, the strong bands at about 33 kDa indicate the expressing of CLEC5A. M = protein marker. NI = Non-Induction. I = Induction.
Figure 13. Western blot of CLEC5A. An anti-His tag was used to bind to the His tag on the CLEC5A protein. The stronger band on the induction lanes suggests the experiments were successful. NI = Non-Induction. I = Induction.
Finally, we expressed CLEC5A on a large scale. The bacterial culture was lysed using a French press, then separated with a high-speed centrifuge. Figure 14. indicates CLEC5A is always found in the pellets no matter how long they were induced.
Figure 14. SDS-PAGE of CLEC5A large-scale expression. The protein was induced for 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours, separately. The bacterial solution was lysed with a French press and separated with a high-speed centrifuge. The results indicate CLEC5A is always expressed in the pellet under these conditions. M = protein marker. Sup. = supernatant.
