Part:BBa_K2382006

T7 promoter + Thioredoxin-MSMEG_5998 fusion protein

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 456
  Illegal XhoI site found at 462
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 901
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

This composite part could successfully express the Thioredoxin-MSMEG5998 fusion protein with pSB1C3 in E.coli BL21. It is an enzyme that could degrade aflatoxin with the aid of coenzyme F420. It belongs to the F420H2-dependent reductases family from Mycobacterium Smegmatis. We also call this part the "Synthetic MSMEG5998" in order to make a distinction with the original MSMEG_5998 from Australia.

Characterization of the Thioredoxin-MSMEG_5998 fusion protein

Expression results

This part expresses the Thioredoxing-MSMEG_5998 fusion protein(BBa K2382009), a specially designed aflatoxin degrading enzyme. In the following description, we will call this fusion protein (BBa K2382009)"Synthetic MSMEG5998", and MSMEG_5998(BBa K2382001) will be called "Australian MSMEG_5998", since the Australian MSMEG_5998 was requested from Taylor, M.C. in CSIRO .from Australia.

IPTG induction

This composite parts (BBa_K2382006) were synthesized by Allbio Life Co., Ltd and put into the standard backbone pSB1C3. First, we transformed the plasmids into E. coli BL21 (DE3) strain to express our proteins. Then IPTG was used to induce the expression system since all plasmids in our project had T7 promoter. We sonicated E. coli and did 9500 rpm and 13000 rpm centrifugation to remove the cell pellet and obtain the supernatant. To confirm the suitable concentration of cell supernatant, we did SDS-PAGE electrophoresis and coomassie brilliant blue staining. The results is demonstrated in the Fig. 1. After centrifuging two times, we could find a high percentage of proteins in the cell supernatant (the 13000 Su group)

Figure 1: Cell lysates were analyzed by SDS-PAGE and coomassie brilliant blue staining. 9500 T meant the initial sample obtained after sonication; 9500 P and 13000 T meant the pellet and the supernatant gotten after 9500 rpm for 20 min; 13000 P and 13000 Su meant the pellet and the supernatant obtained after 13000 rpm for 20 min. (A)(B) Samples contained Australian and synthetic MSMEG5998.

Protein purification, and dialysis

After extracting the cell lysates, we used nickel-resin column to purify our target proteins from the cell lysates because all of our proteins were tagged with 6 histidines at their C-terminal ends. After protein purification, protein dialysis with diaysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH=7.5), and 20% glycerol to remove imidazole in our purified proteins, we did SDS-PAGE gel electrophoresis to ensure our target proteins were successfully purified (Fig. 2A ). The molecular weights of these proteins are listed in the Table 1. The standard BSA proteins were used to quantify the concentration of target proteins.

Table 1: Two expressed recombinant proteins and their molecular weights are listed.

Proteins	Molecular weight
Synthetic MSMEG5998	32.4 kDa
Synthetic FGD	51.5 kDa

Figure 2B: Concentration of proteins was quantified by SDS-PAGE and standard BSA samples with 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 mg/ml. (B) Two recombinant proteins expressed by the plasmids from DNA synthesis.

Protein solubility analysis

To know whether the solubility of our two enzymes (MSMEG_5998 and FGD BBa_K2382002) increased after fusing enzymes with thioredoxin, we dissolved all cell lysates which containing pellet and supernatant and did western blot to detect the content of our target proteins. All proteins were detected by anti-6x His Tag antibody because all of them contained a 6-histidines tail when bacteria expressed them. In Fig. 3, we could find there was good expression of both Australian and synthetic MSMEG5998 in the “13000 Su” group when compared with the “13000 P” group. This result meant that most proteins were dissolved in the supernatant while few proteins deposited in the cell pellet after 13000-rpm centrifugation. However, we could not observe good solubility in both Australian and synthetic FGD because there were little or no difference between the “13000 Su” group and the “13000 P” group.

Figure 3: Cell lysates in the process of two times centrifuge were analyzed by western blot. The abbreviations of five groups were the same as Fig. 1.

Protein Expression Over Time

We transformed the plasmids that contained this part (BBa_K2382006) into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours. Then we use Western Blot mehtod to amalyze the quantaty of Thioredoxin-MSMEG_5998 (Synthetic MSMEG_5998) at each time spot.

Discussion

Figure 4 : The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.

According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the amount of Synthetic MSMEG_5998 were still increasing.

Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.

Figure 5 : The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.

Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot. It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.

Figure 6 : Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.

Enzyme Function Results

Enzyme Activity Assay

The conditions of reaction to degrade aflatoxin by MSMEG5998 were modified from Taylor’s study[3]. All concentrations of reactants are listed in Table 2 and 32 μM aflatoxin was used. We first mixed all reactants in eppendorfs and then put them at 22°C.

Figure 7A: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (A) Direct 365 nm absorbance were detected after mixing Australian/synthetic MSMEG5998 and Australian/synthetic FGD and other reactants at 0th and 8th hour in the environment of pH=7.5 and 22℃.

In Fig. 7A, we compared two proteins, MSMEG5998 and F420-dependent glucose-6-phosphate dehydrogenase (FGD) expressed from Taylor’s vectors (from Australia) and from our synthetic vectors. We found that both the Australian and synthetic MSMEG5998 have great activity and degraded aflatoxin B1 by more than 60%. The effect of the synthetic one may be better than the Australian one but there were no statistic significance.

Table 2: The substance concentration of the aflatoxin-degradation reaction. For convenience sake, we called G6P, F420, FGD, and tris buffer as the reactants.

Name	Concentration
Aflatoxin B1	32 or 10 μM
MSMEG5998	0.1 μM

Reactants

Glucose-6-phosphate (G6P)	2.5 mM
F420	5 μM
F420-dependent glucose-6-phosphate dehydrogenase (FGD)	0.225 μM
Tris-HCl (pH=7.5)	25 mM

However, only Australian FGD has activity to reduce F420 into F420H2 and help the reaction. This finding corresponds with our dry lab results. Therefore, we used Australian and synthetic MSMEG5998 and Australian FGD to do the same experiment again to figure out whether the degradation percentage was dependent of time and whether the main reason of degradation was MSMEG5998.

Figure 7B: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (B) The same way as (A) but Australian/synthetic MSMEG5998 and Australian FGD were used and the reaction were detected at 0th, 2nd, 4th, 6th, and 8th hour. a, p < 0.001 compared to the 0th hour of the synthetic MSMEG5998(+) group; b, p < 0.001 compared to the 0th hour of the Australian MSMEG5998(+) group; c, p < 0.001 compared to the same time of the Australian MSMEG5998(+) group.

Figure 7C: MSMEG5998 could significantly degrade aflatoxin at time manner in vitro. (C) The same way as (B) but the degradation percentage were detected by ELISA. Because the initial concentration of aflatoxin (10000 ng/ml) was too high to be detected by the ELISA, we didn’t demonstrate the initial data.

The results were detected by direct 365 nm absorbance (Fig. 7B) and by ELISA (Fig. 7C). We found out that the degradation percentage was time-dependent. The synthetic MSMEG5998 had better activity than Australian MSMEG5998. The former was able to degrade 83% aflatoxin after 8 h while the latter could only degrade 52% aflatoxin.

MSMEG5998 alleviated aflatoxin-induced p53 pathway activation in HepG2

Cells (5×10 5 / 3.5 cm dish) were divided into 5 groups: control group, aflatoxin group (treated with 10 μM), aflatoxin + reactants group (treated with aflatoxin 10 μM and reactants as Table 2), aflatoxin + reactants + MSMEG5998 group (treated with aflatoxin 10 μM, reactants in Table 2 and synthetic MSMEG5998 0.1 μM), and MSMEG5998 group (treated with synthetic MSMEG5998 0.1 μM).

After 24 h treatment, cells were lysed and their protein expression was analyzed by western blot. In Fig. 8, we found that the expression of p-Chk1 (Ser345), p-Chk2 (Thr68), p-p53 (Ser20), p53, and p21 were all decreased by MSMEG5998 and other reactants when compared to the aflatoxin alone group. This result may attribute to the highly desirable activity of MSMEG5998 of degrading aflatoxin and preventing the toxin from entering cells. </p> Unexpectedly, we also observed a lower expression of these proteins in the aflatoxin + reactants group, which meant that MSMEG5998 may not be the only factor that inhibit the activation of p53 pathway in HepG2 cells. Besides, to ensure that our enzyme MSMEG5998 would not be toxic to cells, we designed a group of MSMEG5998 alone and found that it resembles expression of these proteins as the control group. The results showed that this enzyme was safe.

Figure 8: MSMEG5998 decreased the p53 pathway activation induced by aflatoxin in HepG2. p-Chk1, p-Chk2, p-p53 (Ser20), p53, and p21 were detected by western blot after HepG2 cells (5×10 5 cells/3.5 cm dish) were treated with aflatoxin for 24 h. β-actin was a loading control. All data were standardized with the control group. C: control group; AF: aflatoxin group; A+R: aflatoxin + reactants group: aflatoxin + reactants + MSMEG5998 group; 5998: MSMEG5998 group.

References

(1)Taylor, M.C., et al., Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradation. Molecular microbiology, 2010. 78(3): p. 561-575.

(2)Lapalikar, G.V., et al., F420H2-dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the Actinomycetales. PLoS One, 2012. 7(2): p. e30114.

(3)Creppy, E.E., Update of survey, regulation and toxic effects of mycotoxins in Europe. Toxicology letters, 2002. 127(1): p. 19-28.