Part:BBa_K5137500
Target component (alternatively named as pYY8aTV1)
General Biology
This composite protein is the main component for developing our advanced targeted therapy. A tumor-homing peptide enables the selective targeting of tumor cells, ensuring therapeutic drugs reach cancerous tissues with high specificity [1]. pH-dependent membrane-active peptides allow for controlled membrane disruption in the acidic environments of endosomes, activating only under these conditions. To enhance intracellular delivery, an endosomal protease cleavage site enables the construct to escape from endosomes and avoid degradation[2]. Additionally, mfp1 facilitates liquid-liquid phase separation (LLPS) when incubated with our drug component[3], aiding in the aggregation of molecules, which can improve the efficacy of drug delivery within cells. Together, these elements create a robust system for precise intracellular drug delivery, improving the stability and efficiency of targeted treatments.
Cloning Stategy
Overview
We utilized the Golden Gate assembly method to construct this composite part. This method allows for the directional and simultaneous assembly of multiple DNA fragments in a single reaction. By designing compatible overhangs at the ends of each fragment, we ensured the correct assembly of our tumor-homing peptide, pH-dependent membrane-active peptide, endosomal protease cleavage site, SMT3 and mfp1 into the final composite construct (Figure 1). The efficiency and accuracy of Golden Gate assembly made it an ideal choice for assembling our complex multi-part design.
Amplification of Inserts
To amplify the individual inserts for our composite part, we employed the PCR method. All of our gene fragments, including the tumor-homing peptide, pH-dependent membrane-active peptide, endosomal protease cleavage site, and mfp1, was amplified using custom-designed primers. We also amplified our backbone to insert the overhang. These primers were specifically designed to include desired overhangs compatible with the Golden Gate assembly system. The overhangs ensured that each fragment would be inserted into the vector in the correct orientation and position during assembly. By optimizing the PCR conditions and verifying the product sizes, we successfully generated the necessary inserts with precise overhangs, enabling seamless integration of the components into the final construct (Figure 2).
Primer sequence for amplifying p160 THP, PMAP, EPCS Targeting Part – F: AAG GTC TCA TAT GGT CCC ATG GTT AGA GCC AGC CTA CCA ACG GTT CCT C Targeting Part – R: AAG GTC TCA CCA TGA CAC CAG CGA ACC GTT TAT GAC GGC GAC TGG ATG C Primer sequence for amplifying pYY8a as backbone for Target component construction pYY8a-Plasmid-F: AAG GTC TCA ATG GGA TCG GAC TCA GAA GTC AAT CAA GAA GCT AAG pYY8a-Plasmid-R: AAG GTC TCA CAT ATA TAT CTC CTT CTT AAA GTT AAA CAA AAT TAT TTC TAG AGG GGA ATT
Golden Gate Assembly
Following amplification, the insert and backbone were combined in a single reaction. The Golden Gate assembly relies on type IIS restriction enzymes that cut outside of their recognition sites, allowing for precise ligation of inserts into the backbone. In our experiment, we used Thermo Scientific™ FastDigest Eco31I for restriction digestion and Invitrogen™ T4 DNA Ligase for ligation. All reaction reagent and DNA were mixed by pipetting and incubated at 37°C for 2 hours.
Bacterial transformation
The assembled construct was then transformed into E. coli NEB Stable Competent Cell via heat shock method. The cells were thawed on ice and homogenized by tapping before adding the DNA. 1 μL of plasmid DNA was added to the cell suspension and carefully mixed by flicking the tube 4-5 times and incubated on ice for 30 minutes. Heat shock at 42°C was introduced for 45 seconds and the cells were placed on ice for 5 minutes. The bacteria were recovered by adding 950 μL NEB 10-beta/Stable Outgrowth Medium into the mixture and placed at 37°C shaker incubator with rigorous shaking (250 rpm) for 1 hour. The bacteria were spread on antibiotic plate and incubated at 37°C for overnight.
Verification of the insert sequence
We employed colony PCR and Sequencing techiques to confirm successful integration of the insert into the plasmid backbone after successful bacterial transformation. We sequenced our plasmid on Illumina platform. The plasmid sequence was assembled from fastq file. The assembled sequence was then aligned with reference plasmid sequence and identity was 100%.
Expression and validation of the protein
Expression of the protein
The Target component protein was expressed in E. coli BL21. The gene encoding the protein was cloned under the control of T7 promoter. The bacteria were cultured in LB broth supplemented with Kanamycin (50 µg/ml). The bacteria were cultured at 37°C for 4 hours without any inducer. After the OD600 reached 0.6, IPTG was added and incubated for additional 20 hours.
Testing LB and TB as production culture media
The crude cell lysate from each media was analyzed by running the sample in SDS-PAGE. For better resolution, 10% resolving gel was used. The gel was run at 200 V and 400 mA for 60 min. The gel was stained with Coomassie blue and then destained twice to ensure low blue background. The band on the confirms the presence of the protein (Figure 3).
FPLC Protein Purification and Concentrator
Since eGFP plasmids are being expressed more in TB media, we continued with large-scale protein production in 500 mL TB media and used the pellet for lysis and protein purification. Following FPLC with ÄKTA Go, the fractions from wash and elution steps were screened with 2 x 10% SDS PAGE gel (Figure 4). The target protein band (~29.7 kDa) is visible on the gel despite having some contaminations with other proteins.
To test the LLPS formation from this protein, it is important to concentrate the band using Pierce Protein Concentrators PES with 10 kDa cutoff. The samples with detectable 29.7 kDa band were loaded and centrifuged at 2,300 x g, 4°C until the total volume reached 1 mL. Concentrated protein was desalted in the same column by adding 19 mL 10 mM Tris-HCl and centrifuged with the same speed and temperature until the volume is back to 1 mL. Verification is being conducted with another SDS PAGE gel and the band-of-interest appears more clearly (Figure 5).
LLPS formation through fluorescence microscope
Final test for our target component would be the LLPS formation with Dox-conjugated drug components. Required components to form LLPS were combined and mixed in 0.2 mL PCR tube with the order:
- 2 uL Doxorubicin-conjugated CfaC-CGG8 - 2 uL modified Adf3 as drug component - 4 uL modified Mfp1 as target component
Small aliquot (2 uL) was pipetted to 0.17 mm thin rectangular cover glass (25.4 mm × 76.2 mm) as object slide and fixed by pressing round-shaped cover glass on it. Following that, the slide was mounted and visualized by Axiovert 5 fluorescence microscope with GFP wavelength (±480 nm excitation wavelength) for fluorescent picture. Bright field image was also captured on the same location for discriminating doxorubicin-negative and positive LLPS.
References
- Tong, S. et al. (2022) ‘A small peptide increases drug delivery in human melanoma cells’, Pharmaceutics, 14(5), p. 1036. doi:10.3390/pharmaceutics14051036.
- Yu, S. et al. (2021) ‘Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo’, Nature Communications, 12(1). doi:10.1038/s41467-021-25448-z.
- Yin, Y., Roas‐Escalona, N. and Linder, M.B. (2024) ‘Molecular engineering of a spider silk and mussel foot hybrid protein gives a strong and tough biomimetic adhesive’, Advanced Materials Interfaces, 11(8). doi:10.1002/admi.202300934.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 413
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 413
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 413
Illegal BglII site found at 302 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 413
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 413
- 1000COMPATIBLE WITH RFC[1000]
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