Part:BBa_K5322002
Constitutive Mfp5 Expression System
Contents
Usage and Biology
The plasmid pET29a-J23119-RBS-Mfp5-T7 utilizes the pET29a vector for high-level expression in Escherichia coli. This system is controlled by the strong constitutive promoter J23119, which regulates the expression of the mussel foot protein Mfp5. The ribosome binding site (RBS) ensures efficient translation of the mRNA, while the T7 terminator provides a clean and efficient termination for transcription. This system is designed for the effective expression of Mfp5 under conditions not influenced by environmental factors, allowing it to exhibit its adhesive properties.
Construction of the plasmid
To express eukaryotic proteins in prokaryotic systems, we chose Escherichia coli BL21(DE3) as the chassis cell, using the strong promoter J23119 as the regulatory element for efficient expression of the adhesive protein. As shown in Figure 2-1, we designed the plasmid pET29a-J23119-RBS-Mfp5-T7. The plasmid was transferred to BL21(DE3) via homologous recombination, and single colonies of E. coli were selected on several transformation plates for plasmid extraction. We performed PCR verification using specific primers targeting a 512 bp fragment, as illustrated in Figure 2-2. Plasmids with correctly positioned bands were sequenced, and the sequencing results in Figure 2-3 confirmed the successful construction of the plasmid pET29a-J23119-RBS-Mfp5-T7.
Figure 2-1 Plasmid pET29a-J23119-RBS-Mfp5-T7
Figure 2-2 Colony PCR gel electrophoresis of plasmid pET29a-J23119-RBS-Mfp5-T7(512bp)
Figure 2-3 Sequencing results of plasmid pET29a-J23119-RBS-Mfp5-T7
Protein Expression Validation
We characterized the protein through Tricine-SDS-PAGE and Western Blot experiments, confirming the expression of Mfp5,as shown in Figures 3-1 and 3-2. The size of J23119-Mfp3, including the His tag, is 9.8 kDa; however, based on the results from the gel and Western Blot, we observed a shift in the bands, which approximately appeared around the 13 kDa position. We hypothesize that the strong adhesive properties of Mfp5 may have slowed the protein migration rate, resulting in an upward shift of the band position and an apparent increase in the displayed protein size.
Figure 3-1 Tricine-SDS-PAGE analysis of Mfp5
Figure 3-2 Western Blot analysis of Mfp5
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 300 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 137
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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