Difference between revisions of "Part:BBa K4632003"

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Cowpea trypsin inhibitor(CPTI)

Description

1. How does it work? CPTI can inhibite the serine protease by interacting with the active site of serine protease. Simultaneously, CPTI inhibits insect feeding by stimulating feedback signals from insect (Xuhong-lin et al.,2008)

2. Eco-friendly and Safe CPTI, a member of the Bowman-Birk protein family. Anti-insect spectrum tests show that CPTI inhibits almost all tested major agricultural pests. (Xuhong-lin et al.,2008) And, most importantly. it is Eco-friendly and Safe. (see more detail on[1]）

3. What we have done? (SCAU-China 2023)

The proteinase inhibitor CPTI was chosen, and OmpA was selected as the signal peptide. OmpA has been successfully employed for extracellular expression in prokaryotic systems during laboratory work (Pechsrichuang et al., 2016, Movva et al., 1980). We also obtained information from the Peking University iGEM team, indicating their successful experience using OmpA as a signal peptide (see HP Section). Therefore, using OmpA as a signal peptide for CPTI expression appeared to be a more promising approach (Figure 1)

Figure 1 Diagram of CPTI circuit design

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Construction and Characterization

1. Verifying the Expression and Secretion Proficiency of CPTI

We ordered the OMPA-CPTI fragment from GUANGZHOU IGE BIOTECHNOLOGY LTD and inserted it into the pET30a vector. [2] Then, we transformed pET30a-OMPA-CPTI into E. coli BL21, induced expression with IPTG for 3 hours, and then performed centrifugation to obtain the supernatant and pellet fractions.

Figure1: Diagram of CPTI circuit design

The supernatant was concentrated using ultrafiltration centrifuge tubes, while the pellet was subjected to disruption with lysis buffer. After disruption, we centrifuged the sample to separate the supernatant from the disrupted pellet, and the disrupted pellet was resuspended in lysis buffer.

Since 12% SDS-PAGE did not adequately display the expression results, we also attempted Tricine-PAGE and increased the SDS-PAGE separation gel concentration to 17.5%. However, upon analyzing these samples using Tricine-PAGE and Western Blot, the results revealed that the CPTI protein was not expressed in E. coli (as shown in Figures 2 and 3).

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Figure 2: Tricine-PAGE analysis of CPTI expression Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysateof of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)

Figure 3: Western blot analysis of CPTI expression Lane 1：Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane4:Whole cell lysate of OmpA-CPTI (+IPTG);Lane5:Whole cell lysate of OmpA-CPTI (-IPTG);Lane6:Whole cell lysate of CPTI (+IPTG);Lane7:Whole cell lysate of of CPTI(-IPTG);Lane8:Whole cell lysate of pET-30a(+IPTG);Lane9:Whole cell lysate of pET-30a(-IPTG)

In conclusion, the expression of the CPTI protein in E. coli was unsuccessful as observed in the results from both 12% SDS-PAGE, Tricine-PAGE, and 17.5% SDS-PAGE. Further investigation and optimization may be necessary to achieve successful protein expression.

Subsequently, we have tried to constructed the CPTI gene with a GST tag[3], and protein induction expression is currently underway. This modification is intended to ensure that the protein is expressed to a significant extent. Previous literature has demonstrated a similar approach, and the key difference in our attempt is to test the activity of CPTI without removing the GST tag.

2. Measurement of CPTI Activity

The proteinase inhibitor failed to express successfully, as the intended proteinase inhibitor was a pancreatic trypsin inhibitor. The original plan was to determine the activity of the expressed proteinase inhibitor using the BAEE method, the principle of which is explained below:

The principle of the BAEE (Nα-Benzoyl-L-arginine ethyl ester hydrochloride) assay for measuring trypsin inhibitor (TI) activity is based on the ability of a pancreatic trypsin inhibitor to bind with pancreatic trypsin. BAEE is a substrate catalyzed by pancreatic trypsin, and the product of the catalytic reaction by pancreatic trypsin with BAEE absorbs light at a wavelength of 253 nm. The rate of increase in absorbance at A253 per unit time (min) represents the activity of pancreatic trypsin (U1). When pancreatic trypsin inhibitor (TI) is added to the reaction, TI inhibits the rate of production of the product in the BAEE reaction catalyzed by pancreatic trypsin, resulting in a slower rate of increase in absorbance at A253 per unit time (min). This reduced rate represents the residual activity of pancreatic trypsin after inhibition by TI (U2). Therefore, the activity of TI (TIA) can be calculated as follows: TIA = U1 - U2.

The activity of pancreatic trypsin (U1) is calculated as follows:

U1 = ΔA253/t / 0.001

The activity of pancreatic trypsin after inhibition by TI (U2) is calculated as follows:

U2 = ΔA253/t / 0.001

The activity of TI (TIA) can then be determined as:

TIA = U1 - U2

References

Xuhong-lin, Zhaihong-li, Wangfeng et al. Cowpea Trypsin Inhibitor Gene(cpti) and its Application in Insect Resistance Transgenic plants[J]. 2008.

Wei Gao, Feng Li, Qingyu Lu. CPTI (Cowpea Trypsin Inhibitor) gene, applications thereof and method for preparing great amount of CPTI: CN102127552B[P]. 2013-02-13.

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 <p><strong> 1. Verifying the Expression and Secretion Proficiency of CPTI</strong></p>