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Applications of BBa_K4632003

1. Verifying the Expression and Secretion Proficiency of CPTI

The OmpA -CPTI fragment (ordered from Guangzhou IGE Biotechnology Co.,Ltd.) was inserted into the pET-30a vector to generate the plasmid pET-30a-OmpA-CPTI. This plasmid was then transformed into E. coli BL21 and cultured overnight in LB medium. Overnight culture was dilute by 1/1000 to fresh LB medium, the IPTG was added when the OD600 reach 0.6. The medium was incubated another 3 hours after the ITPG addition, then centrifuged at 12,000 rpm 10 mins to obtain the supernatant and the precipitate. The supernatant was concentrated using ultrafiltration centrifuge tubes, while the precipitate was subjected to cell lysis with a lysis buffer. After lysis, the sample was centrifuged again to separate the supernatant from the post-lysis precipitate. The post-lysis precipitate was resuspended in lysis buffer. Tricine-PAGE (Figure 3) and Western Blot analyses (Figure 4) were performed on the above samples, and the results indicated that the CPTI protein was not expressed in E. coli.

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Figure 3. Tricine-PAGE analysis of CPTI expression. <p>Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysateof of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

Figure 4: Western blot analysis of CPTI expression <p>Lane 1: Concentrated supernatant of OmpA-CPTI (+IPTG); Lane 2: Concentrated supernatant of OmpA-CPTI (-IPTG); Lane 3: Concentrated supernatant of pET-30a(+IPTG); Lane 4: Whole cell lysate of OmpA-CPTI (+IPTG); Lane 5: Whole cell lysate of OmpA-CPTI (-IPTG); Lane 6: Whole cell lysate of CPTI (+IPTG); Lane 7: Whole cell lysate of of CPTI (-IPTG); Lane 8: Whole cell lysate of pET-30a (+IPTG); Lane 9: Whole cell lysate of pET-30a (-IPTG)

With IPTG the cell could produce the detectable CPTI (Lane 4, Figure 4), however, the concentrated supernatant of CPTI could not been seen (Lane 1, Figure 4). This might because of the poor solution of CPTI with OmpA singlal peptide. To get a extracellular expressed CPTI, the GST tag, which could improve the solution of certain protein, was successfully added at the C-terminal of the CPTI gene. The protein induction expression experiment is currently underway.

This modification ensures that the protein can be expressed to a significant extent, as similar experiments have been reported (Yang et al., 2003). Our unique approach involves testing the activity of CPTI without removing the GST tag, which distinguishes our study from previous ones.

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. [1] 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[2], 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.

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