Cycle 1: Detecting the expression of color-removing laccases
Design
As we aim to achieve the removal of dyes, we chose laccase as our protein for color removal. In our initial
design, laccase includes four different types, namely Peer J, Glac 1, Lcc3, bpul, and a segment of rk sequence
is also translocated into the plasmid to stabilize NGCatcher.
Build
We introduced these laccases into NGCatcher, separately transformed into four different plasmids. The reason
for using only NGCatcher is that we had not designed additional protein functional sequences in the first cycle.
We added a His-Tag purification system at the end of each sequence, and protein expression was induced using
IPTG.
Test
SDS-PAGE analysis revealed that the translocated sequences were not successfully expressed. After running the
gel, we found that the purification effect was not good. And our protein did not appear in the expected band.
Learn
During the purification process, the protein was quickly eluted, so the salt concentration in the elution
buffer needed to be adjusted downward for better protein separation. (Fig.1) (Fig.2)
Fig. 1 Before improvement
Fig. 2 After improvement
Cycle 2: Detecting the function of hydrophobin
Design
In the research process, we discovered that color catchers on the market are one of the solutions to dye
problems. However, existing products merely capture the dye on the color catcher without breaking it down.
Hence, we thought of combining laccase and NGCatcher. To bring the target protein closer to the surface of our
color catcher, we used hydrophobin in the sequence. Hydrophobin can alter the properties of the color catcher
surface, increase its hydrophilicity, and it carries a fluorescent sequence.
We fused hydrophobin with NGTag and finally used NGCatcher/Tag system to capture laccase and hydrophobin
together.
Build
Initially, we translocated HFB9b(hydrophobin) in the sequence and added His-Tag at the end. We applied
hydrophobin to the color catcher, immersed it in a stagnant water tank, and tested whether the water contained
fluorescent substances to determine if hydrophobin was attached to the color catcher.
Test
Protein expression was observed through SDS-PAGE, and we observed protein bands, but they were not in the
expected positions. To test its activity, we first attached hydrophobin to the desired surface, then dropped
water to measure its contact angle. A smaller contact angle indicates greater hydrophilicity.
Learn
Referring to other literature, we found that expressing proteins through E. coli produces non-functional
inclusion bodies, which affect the expression of the target protein's activity. Removal of inclusion
bodies
is
necessary to restore activity. Therefore, we found the DewA series of hydrophobin, as it has been reported to
induce the production of inclusion bodies.
Reference
Soluble Expression and Efficient Purification
of
Recombinant Class I Hydrophobin DewA
