Inverse Fusion PCR
We designed primers to insert a linker and alpha factor on the carboxyl termini of our encapsulin protein, which is known to be on the protein's exterior surface. This was done to facilitate endocytosis in yeast. To ensure unique binding of our cloning primers, we added alpha factor at the end of a 6 residue glycine-alanine linker. Retrospectively, we realized that this sequence seems a bit hydrophobic to serve as a good linker, however, it is surrounded by hydrophilic alpha factor and spytag which may mitigate its hydrophobic tendencies.
Primer design was based on a published protocol for inverse fusion PCR (citation). Three primers are required for this procedure: a forward primer complementary to vector nucleotides immediately following the desired insertion site, a reverse primer complementary to vector nucleotides before the insertion point and containing the insertion sequence, and a second reverse primer complementary to the end of the insert.
This was designed to allow two reactions in the same pcr experiment. First, the primers with forward and reverse complements to the vector insert add the insertion sequence to the vector and linearize it. Second, the forward primer and the second reverse primer amplify the vector-insert fusion. Finally, the phosphorylated forward primer allows ligation of the fused sequence.
All primers were designed with a melting temperature of ~58 degrees, as recommended in the literature and were checked with IDT oligo analyzer for hairpins, homodimers, and heterodimers. Unique binding was confirmed with Snapgene.
Repeated efforts to implement inverse fusion pcr were unsuccessful, likely due to incorrect primer concentrations in PCR.
Encapsulin Protein Purification
Using encapsulins as a cell type specific drug delivery platform requires their endocytosis to be regulated by an engineered ligand. To show that the encapsulin is not universally endocytosed in the absence of an added ligand, we purified bare encapsulins, lacking the added alpha factor ligand.
The protein was successfully expressed in BL21 E. Coli, and purified with a nickel column via a his-tag on the exterior of the encapsulin. The successful purification demonstrates the self assembly of the encapsulin loaded with protein cargo, showing the ease of encapsulin creation for protein-based therapeutics.
SDS Page Gel confirmed successful purification. This was evaluated based on the expected size of the encapsulin, which was approximately 37 kDa.
Figure 1: SDS Page Results
Further fluorescent microscopy showed possible encapsulin clusters.
Figure 2: Fluorescence Microscopy (possible encapsulin clusters circled in white)
Protein concentration was measured with a NanoDrop to be on the order of 3 ng/ul.
Encapsulin Endocytosis
An endocytosis protocol was run on the encapsulin (without alpha factor) into yeast cells. The purpose of this experiment was to serve as a negative control in which endocytosis fails to occur without the alpha factor present to mediate endocytosis. The following images show pure yeast and yeast with the encapsulin added. While it’s possible that endocytosis failed, the images themselves make a definitive conclusion difficult to make.
Figure 3: Pure Yeast
Figure 4: Yeast + Encapsulin