Part:BBa_K5137502

Drug payload (alternatively named as pCfaC-CGG8)

General Biology

This composite protein is the drug payload component for developing our advanced targeted therapy. The CfaC domain plays a crucial role in ligating the recombinant protein to the drug component^[1], acting as a linker that connects the therapeutic payload to the delivery system. Additionally, the CGG repeat sequence is designed specifically for drug conjugation, with the cysteine residue within the CGG repeat serving as a reactive site for covalent attachment to the drug molecule. This strategic inclusion of ligation and conjugation motifs ensures precise binding and delivery of the drug, offering a powerful tool for controlled and targeted therapeutic applications.

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 CfaC-CGG8 insert 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.

Figure 1: Schematic diagram of Drug payload construction

Amplification of Inserts

To amplify the individual inserts for our composite part, we employed the PCR method. Our gene fragment, CfaC-CGG8 was amplified using custom-designed primers. Homology arms from insert fragment also ensuring the amplification viability without having miss annealed primer at CGG8 region. 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 CfaC, CGG8 
CfaC- CGG8 v2 F: GAG CGG ATA ACA ATT CCC CTC TAG AAA TAA TTT TGT TTA ACT TTA AGA AGG AGA TAT ATA 
CfaC- CGG8 v2 R: CAG CTT CCT TTC GGG CTT TGT TAG CAG CCG GAT CTC A 

Figure 2: Workflow of construction of Target component

Gibson Assembly

Following amplification, DNA concentrations for CfaC-CGG8 fragment was measured with Nanodrop. Required DNA amount to do Gibson Assembly with 3:1 ratio of insert:vector (pYY5a) was prepared based on calculations through NEBioCalculator® (https://nebiocalculator.neb.com/#!/ligation). Mix all components, including GeneArt™ Gibson Assembly® HiFi Master Mix by pipetting and incubate in PCR machine at 50°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 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 24 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).

Figure 3: Protein bands on SDS-PAGE from 2 production media and OD600 measurement

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 SDS PAGE gel. The results are shown on Figure 4.

Figure 4: Fractions screening for pCfaC-CGG8 FPLC result

The results indicated multiple protein bands across elution step. Coincidentally, the molecular weight of some bands are matching the conditions if cysteine residue from CGG8 region forms unspecific disulfide bond with other CfaC-CGG8 chain. Still, the sample was processed through Pierce Protein Concentrator PES, 10 kDa cut-off. All suspected-multimer CfaC-CGG8 appear more intense in verification gel (Figure 5).

Figure 5: Fractions screening for pCfaC-CGG8 FPLC result

Intein Functionality Test

First, we tested the functionality of intein splicing activity by incubating CfaC-CGG8 with eGFP-CfaN for 30 minutes at room temperature. With the abundance (following FPLC) and higher molecular weight on eGFP counterpart, any intein activity will be resulted in appearance of higher band. Through SDS-PAGE, we noticed the appearance of new protein band with greater intensity and may correspond to 1 eGFP CfaN being ligated with multimeric version of CfaC-CGG8 (Figure 8).

Figure 6: Observable intein activity between CfaC-CGG8 and eGFP-CfaN

LLPS formation through fluorescence microscope

Additionally, fluorescence microscope of Dox-conjugated CfaC-CGG8, combined with other drug and target components, confirmed the presence of fluorescence LLPS (Figure 7). This indicated Doxorubicin has been encapsulated inside the formation following protein ligation.

Figure 7: Fluorescence signal from LLPS with target and Dox-conjugated drug components. Negative control is shown at the bottom row.

References

1. Stevens, A.J. et al. (2016) ‘Design of a split intein with exceptional protein splicing activity’, Journal of the American Chemical Society, 138(7), pp. 2162–2165. doi:10.1021/jacs.5b13528.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 21