Results

The gene fragments TyrBm, TyrBm-L1-CipA, CipA-L1-TyrBm, TyrBm-L2-CipA, and TyrBm-L3-CipA were synthesized by GenScript and cloned into a cloning vector. We amplified the gene fragments from the cloning vector and ligated them into the expression vector pET-21a(+), finally constructing the recombinant plasmids TyrBm_pET-21a(+), TyrBm-L1-CipA_pET-21a(+), CipA-L1-TyrBm_pET-21a(+), TyrBm-L2-CipA_pET-21a(+), and TyrBm-L3-CipA_pET-21a(+).

Since the linker contained restriction enzyme sites, TyrBm_pET-21a(+) was constructed using the double restriction enzyme digestion method. In contrast, TyrBm-L1-CipA_pET-21a(+), CipA-L1-TyrBm_pET-21a(+), TyrBm-L2-CipA_pET-21a(+), and TyrBm-L3-CipA_pET-21a(+) were constructed using the homologous recombination method.

We used the cloning vector containing the target gene as a template and corresponding primers to amplify the target gene. After gene amplification, agarose gel electrophoresis was performed, and the fragments were recovered using a gel extraction kit.

We used the restriction enzymes BamH I and Xho I to digest the TyrBm PCR product and the expression vector pET-21a(+). The digested fragments were recovered using a gel extraction kit.

The digested TyrBm fragment and expression vector pET-21a(+) were ligated using T4 DNA ligase and then transformed into E. coli Top10 competent cells.

The PCR products of TyrBm-L1-CipA, CipA-L1-TyrBm, TyrBm-L2-CipA, and TyrBm-L3-CipA were ligated with the digested expression vector pET-21a(+) using a homologous recombination kit and then transformed into E. coli Top10 competent cells.

After overnight culture, colony PCR verification of single colonies indicated that we obtained positive clones.

We performed bidirectional sequencing on the positive clones using the universal T7 and T7-TER primers from Sangon Biotech. SnapGene analysis results confirmed that the sequences of our constructed plasmids were correct.

The constructed plasmids were transformed into E. coli Rosetta competent cells, and single colonies were selected for amplification.

Protein expression was induced with IPTG. Samples were taken from the cultures before induction and after induction. The cells were washed twice with PB buffer (pH=7.4), then lysed and denatured in protein loading buffer at 96°C, followed by analysis using SDS-PAGE. A small amount of protein expression was observed even before induction, possibly due to background expression or the small culture volume. The protein yield increased significantly after IPTG induction, as shown in Figure 6. The results confirmed successful induction of protein expression for all constructs.

Protein expression was induced overnight at 37°C with 0.5 mM IPTG. After induction, the cells were disrupted using an ultrasonic disruptor, and the supernatant and inclusion bodies were collected separately. Proteins from samples taken before induction, after induction, the supernatant, and the inclusion bodies were analyzed by SDS-PAGE. Protein expression significantly increased after induction, and the protein content in the inclusion bodies was higher than in the supernatant, as shown in Figure 7.

First, we used the tyrosine plate colorimetric method to observe whether the bacterial cultures turned black after cultivation. E. coli transformed with all five plasmids were able to synthesize tyrosinase, causing the plates to turn black, as shown in Figure 8.

Next, we used lysates of disrupted E. coli to catalyze the tyrosine derivatives Boc-L-Tyr-OMe and Boc-L-Tyr. We used lysates from 5 ml of culture for the catalytic reactions. Based on the color intensity of the final solutions, TyrBm-L1-CipA and TyrBm-L2-CipA showed higher enzyme activity.

To quantitatively measure the enzyme activity of the different synthesized proteins, we used L-tyrosine as the substrate and detected the enzyme activity of each protein sample under optimized reaction conditions (containing 0.2 mmol/L CuSO₄, pH 7.0 buffer system). The change in absorbance at 475 nm (A475) was dynamically monitored using a microplate reader, and the initial reaction rate was calculated. One unit of enzyme activity (U) was defined as the amount of enzyme required to produce 1 μmol/L of dopachrome per minute (ΔA475=0.001/min).

Figure 10 shows the enzyme activity curves and calculated enzyme activity units for the five proteins—TyrBm, CipA-L1-TyrBm, TyrBm-L1-CipA, TyrBm-L2-CipA, and TyrBm-L3-CipA—in both the supernatant and inclusion body fractions. The experimental results indicate that among all the constructs, the TyrBm-L1-CipA fusion protein (i.e., with the (Gly₄-Ser)₃ Linker and CipA at the C-terminus) exhibited the highest tyrosinase activity in both the soluble supernatant and inclusion bodies, and was therefore selected as the best candidate protein for subsequent dyeing experiments.

Comparing the detected enzyme activities of TyrBm, CipA-L1-TyrBm, TyrBm-L1-CipA, TyrBm-L2-CipA, and TyrBm-L3-CipA revealed that the enzyme activity of TyrBm-L1-CipA was the highest, both in inclusion bodies and supernatant. Therefore, we selected TyrBm-L1-CipA as the target protein for subsequent dyeing experiments. The dyeing experimental procedure is described in the Experiment section. To enhance color diversity, we selected four substrates: L-tyrosine, and the L-tyrosine derivatives Fmoc-L-Tyr, Boc-L-Tyr, and Boc-L-Tyr-OMe. The dyeing results are shown in Figure 11. With the exception of leather, good dyeing effects were observed on the other fibers.