Envelope Protein (E protein)
We found no colonies of DH5α after the transformation into DH5α and suspected that there might be issues during PCR. One of the reasons could be that an adenine would be added to the 3’ on the blunt end of the E protein sequence during PCR, which could have hindered our polymerase, Taq. Therefore, we replaced Taq with Pfu and obtained colonies of DH5α. However, strong bands were only found at 300 bp in the colony PCR (Figure 1.), suggesting that there are still problems during ligation. Currently, we are trying to find a condition such as using a higher concentration of insert or a vector that is suitable for ligation.
Figure 1. Colony PCR of pET-29a(+)_E protein, which only has a size of about 300 bp rather than 500 bp. M = DNA marker. P =e positive control, pET-29b(+)_CLEC5A.
CLEC5A
Colony PCR
We used colony PCR to confirm whether the transformation of pET-29b(+)_CLEC5A into DH5α was successful. However, the results of colony PCR (Figure 2.) shows that there are no bands between 750 and 1,000 bp. We further verified whether pET-29b(+)_CLEC5A could be digested as expected. Figure 3. shows that digestions of pET-29b(+)_CLEC5A with NcoI and HindIII work well. Therefore, we designed new primers to improve the transformation. The colony PCR suggests the transformation can be done with these new primers (Figure 4.), since the sequence of pET-29b(+)_CLEC5A containing the Myc tag, HA tag, and T7 promoter and terminator has a size of about 900 bp.
Figure 2. Colony PCR of pET-29b(+)_CLEC5A with T7 promoter and terminator, Myc tag, and HA tag using the original primers. The expected strong bands between 750 and 1,000 bp are missing. M = DNA marker.
Figure 3. Digestion of pET-29b(+)_CLEC5A using NcoI and HindIII show strong bands between 500 and 750 bp, suggesting that the digestions were successful. M = DNA marker.
Figure 4. Colony PCR of pET-29b(+)_CLEC5A with the new primer shows strong bands between 750 and 1,000 bp, corresponding to the sequence size of pET-29b(+)_CLEC5A containing a Myc tag and T7 promoter and terminator.
Expression on a Large Scale
We have confirmed the expression of CLEC5A with a small-scale culture using Western blot, so we tried large-scale expression. However, since CLEC5A was only found in the pellet, we tried changing the induction time so CLEC5A would be expressed in the supernatant. However, Figure 5. shows that CLEC5A was still always found only in the pellets. We are currently trying to find a condition in which CLEC5A would be expressed in the supernatant.
Figure 5. The SDS-PAGE of CLEC5A with a large-scale expression. The protein was induced for 30 minutes, 1 hour, 2 hours, 3 hours, or 4 hours. However, the results shows that CLEC5A was always expressed in the pellet under these conditions. M = protein marker. Sup. = supernatant.
