pET28a-UAO, pET28a- a llantoinase and pET28a- allantoicase

We added restriction sites BamHI and XhoI to primer, and further amplified the target gene UAO by PCR. The length of the target gene UAO is 981 bp. Figure 2 shows that the fragment lengths are consistent with the results. We added restriction sites BamHI and NotI to primer, and further amplified the target gene a llantoinase by PCR. The length of the target gene a llantoinase is 1485 bp. Figure 2 shows that the fragment lengths are consistent with the results. We added restriction sites SacI and XhoI to primer, and further amplified the target gene allantoicase by PCR. The length of the target gene allantoicase is 1288 bp. Figure 2 shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene allantoicase, allantoinase and UAO.

Figure 2. Electrophoresis for the target genes UAO, allantoinase and allantoicase

Then, we cut the vector pET28a with restriction endonuclease, and used the uncut vector as a control. The length of the pET28a vector is 5369 bp, and the control group of Figure 3 is correct. Line 1-2 is the band of pET28a cut with BamHI and XhoI. Line 3-4 is the band of pET28a cut with BamHI and NotI. Line 5 is the band of pET28a cut with SacI and XhoI. The results show that the plasmid has been cut into linearization.

Figure 3. The electrophoresis for testing the linearized vector pET28a

Note ：

Line1- 2: The band of pET28a cut with BamHI and XhoI

Line 3- 4: The band of pET28a cut with BamHI and NotI

Line 5: The band of pET28a cut with SacI and xhoI

Next, we connect UAO, a llantoinase and allantoicase with pET28a by T4 ligase, and transformed the plasmid into DH5α. Figure 4A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. The length of the target gene a llantoinase is 1485 bp. The length of the target gene allantoicase is 1288 bp. Figure 4C shows that the fragment lengths are consistent with the results. We have successfully the vector pET28a-UAO, pET28a- a llantoinase and pET28a- allantoicase were transformed into DH5α, and the sequencing results in Figure 4B showed no gene mutation. It indicates that our plasmid has been successfully constructed.

Figure 4. The Electrophoresis of the Monoclonal Antibody in DH5α

Note:

A Culture plates UAO, allantoinase and allantoicase for DH5α

B.Electrophoresis of the Monoclonal Antibody UAO, allantoinase and allantoicase

C.The sequencing result of pET28a-UAO,pET28a- a llantoinase and pET28a-allantoicase

pGex-4T-1-UAO and pGex-4T-1-UAA

We used pET28a-UAO, pET28a-allantoinase and pET28a-allantoicase as templates for the following experiments.

2.1 pGex-4T-1-UAO

We wanted to use homologous recombination for constructing pGex-4T-1-UAO, so we added homologous arms at both ends of the primer, and amplified UAO and pGex-4T-1 by PCR. The length of UAO is 981 bp. Figure 5A shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene UAO. The length of the plasmid pGex-4T-1 is 4328 bp. Figure 5B shows that the pGex-4T-1 band is about 4500 bp. It indicates that we have successfully amplified the plasmid.

Figure 5. The electrophoresis for the target genes UAO and pGEx-4T-1 vectors

Next, we transformed the plasmid pGex-4T-1-UAO into DH5α. Figure 6A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. Figure 6B shows that the UAO band is about 1000 bp. The results show that the vector pGex-4T-1-UAO was transformed into DH5α, and the sequencing results of pGex-4T-1-UAO in Figure 6 CD showed that UAO has no gene mutation. It proves that the plasmid pGex-4T-1-UAO was successfully constructed. It indicates that the plasmid pGex-4T-1-UAO has been successfully constructed.

Figure 6. Culture plates for DH5α and Electrophoresis of the Monoclonal Antibody

Note:

A: Culture plates pGex-4T-1-UAO for DH5α

B: Electrophoresis of the monoclonal Antibody pGex-4T-1- UAO

C and D: The Sequencing results of pGex-4T-1-UAO

2 pGex-4T-1-UAA

We used homologous recombination to construct pGex-4T-1-UAA, and add homologous arms at both ends of the primer and amplified a llantoinase, allantoicase and pGex-4T-1-UAO by PCR. The length of a llantoinase is 1485 bp. The length of the target gene allantoicase is 1288 bp. Figure 7 A shows that tthe fragment lengths are consistent with the results. It indicates that we have successfully amplified the target gene. The length of the plasmid pGex-4T-1-UAO is 5301 bp. Figure 7B shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the plasmid(Figure 7C).

Figure 7. Electrophoresis for the genes a llantoinase, allantoicase and pGex-4T-1-UAO

Next, we transformed the plasmid pGex-4T-1-UAA into DH5α. Figure 8AB shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene allantoicase is 1074 bp. In Figure 8C, line1-5 shows that the fragment lengths are consistent with the results. The a llantoinase is approximately about 1485bp. In Figure 8D, line1,4, 5 shows that the fragment lengths are consistent with the results. It indicates that we have successfully amplified the plasmid pGex-4T-1-UAA.

Figure 8. The electrophoresis of the monoclonal antibody of pGEX-UAA in E.coil DH5α

Afterward, we chose positive isolated colonies 1, 4, and 5 and sent them to the company for sequencing. The results showed that the sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid was normal (Figure 9), without any genetic mutations. It indicates that our plasmid pGex-4T-1-UAA has been successfully constructed.

Figure 9. The sequencing of the allantoicase and allantoinase genes on the pGex-4T-1-UAA plasmid

Protein expression and purification

pGex-4T-1-UAO

1.1pGex-4T-1-UAO(BL21(DE3))

In order to improve the expression of the protein, we first tested the protein in E.coil BL21(DE3). So we transformed the plasmid pGex-4T-1-UAO into E.coil BL21(DE3). Figure 9A shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. Figure 10 B shows that the UAO band is about 1000 bp. The vector pGex-4T-1-UAO was successfully transformed into E.coil BL21(DE3).

Figure 10 . Electrophoresis of the Monoclonal Antibody Validation process for pGex-UAO in BL21(DE3)

In order to improve the expression of UAO protein, we designed different gradients of IPTG induction. Table 1 and Figure 1 1 showed that the protein concentration of pGex-UAO increased first and then decreased with the increase of IPTG concentration. When the concentration of IPTG was 0.5 mmol, the protein concentration of UAO was the highest.

Table1: Protein concentration of UAO (mg/mL)

Figure 1 1 . The relationship between the concentration of IPTG and the concentration of proteins

Next, we induced the protein UAO with 0.5 mmol IPTG. The size of the UAO protein is 40 kDa. Figure 1 2 shows that the crude protein has the target protein UAO, and the purified protein solution also has a small amount of UAO, but the purification loses som e proteins.

Figure1 2 . The pGex-4T-1-UAO of SDS-PAGE in BL21(DE3)

1.2pGex-4T-1-UAO(Nissle 1917)

Finally , we transformed the plasmid pGex-4T-1-UAO into Nissle 1917. Figure 1 3 B shows that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene UAO is 981 bp. Figure 1 3 A shows that the UAO band is about 1000 bp. The vector pGex-4T-1-UAO was successfully transformed into Nissle 1917.

Figure 1 3 . Electrophoresis of the monoclonal antibody for pGex-UAO in Nissle 1917

Meanwhile, we induced the protein UAO with 0.5 mmol IPTG in Nissle 1917. The size of the UAO protein is 40 kDa. Figure 1 4 shows that the crude protein has the target protein UAO. In the control group, Nissle 1917 had no target protein of UAO.

Figure 1 4 . The pGex-4T-1-UAO of SDS-PAGE in Nissle 1917

2. pGex-4T-1-UAA

In order to improve the expression of the protein, we first tested the protein in E.coil BL21(DE3). So we transformed the plasmid pGex-4T-1-UAA into E.coil BL21(DE3). Figure 14A and B show that the isolated colonies were successfully grown, and the isolated colonies were selected for colony verification. The length of the target gene allantoinase is 1485 bp and allantoinase is 1047bp. Figure 1 5 C and 15 D show that the fragment lengths are consistent with the results, we have successfully the vector pGex-4T-1-UAA was transformed into E.coil BL21(DE3).

Figure 1 5 . The electrophoresis of the monoclonal antibody validation process for pGex-UAA in E.coil BL21(DE3)

Note:

Figure 1 5 A-B: The monoclonal plate pGex-UAA in BL21(DE3)

Figure 1 5 C: The Electrophoresis verification of allantoicase in pGex-UAA (BL21(DE3))

Figure 1 5 d: The Electrophoresis verification of allantoinase in pGex-UAA (BL21(DE3))

In order to improve the expression of UAA protein, we designed different gradients of IPTG induction. The size of the UAA protein is 118.6 kDa. Figure 1 6 shows that the crude protein has the target protein UAA. However, 0.1mmol, 0.25 mmol, 0.5mmol,0.7mmol and 1mmol IPTG had no significant effect on the expression of UAA protein.

Figure 1 6 . The pGex-4T-1-UAA of SDS-PAGE in (BL21(DE3))

Enzyme activity detection

pGex-4T-1-UAO

A.The Standard Curve

Firstly, we made a standard curve for the determination of uric acid. The second table is the original data of the table. We use a linear function to fit. Figure 1 7 shows that the equation y = 1.1529x + 0.2129 is satisfied, and the correlation coefficient is greater than 0.99. The table fits well(Table 1).

Table 1. Result and curve for the Standard Curve

Figure1 7 . The Standard Curve of Urate Oxidase

The activity of urate oxidase

In order to determine the ability of our constructed plasmid pGex-4T-1-UAO to decompose uric acid, we used uric acid as a substrate and added UAO protein. Table 2 and Figure 1 8 showed that with the increase of UAO protein concentration, the content of uric acid gradually decreased, indicating that UAO is active and can successfully decompose uric acid. And we used E.coil BL21 ( DE3 ) as the control group, the uric acid content of the control group had no obvious change trend.

Table 2. The activity of urate oxidase

Figure 1 8 . The curve of UAO activity

pGex-4T-1-UAA

A. The activity of urate oxidase

In order to determine the ability of our constructed plasmid pGex-4T-1-UAA to decompose uric acid, we used uric acid as a substrate and added UAO protein. Table 3 and Figure 1 9 showed that with the increase of UAA protein concentration, the content of uric acid gradually decreased, indicating that UAA is active and can successfully decompose uric acid. And we used E.coil BL21 ( DE3 ) as the control group, the uric acid content of the control group had no obvious change trend.

Table 3. The activity urate oxidase

Figure 19 . The curve of UAA activity

Contrast enzyme activity of UAO and UAA

By comparing the levels of uric acid after adding UAA and UAO proteins(Figure 20 ), it was found that the uric acid content was lower with UAA compared to UAO. This suggests that UAA has slightly higher enzymatic activity than UAO, further indicating that UAA has a stronger ability to degrade uric acid. However, the protein expression level of UAA can be optimized in order to enhance its enzymatic activity in subsequent experiments.

Figure 20 . The curve of UAA and UAO activity

Future plans

In future studies, we can optimize the protein expression levels of urate oxidase (UAO), allantoinase, and allantoicase. We will further test the protein expression and enzyme activity of pGEX-4T-1-UAA in Nissle 1917 to find the optimal expression conditions. We also need to validate the ability of urate oxidase (UAO), allantoinase, and allantoicase to degrade uric acid in large quantities. Additionally, it is worth considering how to enhance their degradation capabilities. Apart from that, the role of urate oxidase in degrading uric acid in the body is still pending verification. The safety of probiotics also requires further validation.

Reference

1. T. Neogi, Clinical practice. Gout. N Engl J Med 364 , 443-452 (2011).

2. Y. Zhu, B. J. Pandya, H. K. Choi, Prevalence of gout and hyperuricemia in the US general population: the National Health and Nutrition Examination Survey 2007-2008. Arthritis Rheum 63 , 3136-3141 (2011).