Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

Abstract

In vivoimaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable 'cargo' , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher byE. coli,however withCaulobacter crescentuscells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targetedin vivoimaging.

Keywords: covalent chemistry; protein materials; spyCatcher; super-resolution imaging.

Figure 1.

Design, construction and purification of CPP-SpyCatcher fusion proteins. (A) Diagram of hypothesized function of CPP-SpyCatcher fusion proteins. The CPP domain of these constructs would allow binding and penetration across the membrane of gram negative bacteria as well as other organisms. OM-outer membrane; IM-inner membrane. (B) Design of the CPP-SpyCatcher fusion. CPP sequences were placed between the TEV cleavage site in SpyCatcher003 and a GGGS spacer sequence. (C) Purification of CUPID-SpyCatcher003. Lane 2: HisTap HP elution, Lane 3: SEC (Superdex200 16_600), Lane 4: monomeric Hisx6-CUPID-SC + TEV Protease (incubated 16 h), Lane 5: cleaved CUPID-SC, Lane 6: TEV and mixture of cleaved and uncleaved CUPID-SC from NiNTA elution,. (D) Penetratin-SpyCatcher. Lane 2: Hisx6-Penetratin-SC purified by HisTap HP, Lanes 3-5 (further purification by SEC), Lane 3: multimerized fraction, Lane 4: dimeric fraction, Lane 5: monomeric fraction, Lane 6: cleaved Penetratin-SC.

Figure 2.

Size exclusion chromatography of CPP-SpyCatcher constructs shows dimerization and multimers. The CPP-SpyCatcher003 constructs were analyzed by SEC. SpyCatcher003 with a S49C mutation appear mainly as monomers in solution. The cysteine mutation appears to induce some dimerization. Addition of CPP sequences to the N-terminus of SpyCatcher003 induced dimers or high order multimers. Molecular weights were determined by running SEC standards. Elution volumes were normalized by dividing the column volume and are reported as R _f.

Figure 3.

Functional assay of CPP-SpyCatcher bonding to SpyTag: SpyTag-MBP (STMBP) added to each SpyCatcher construct at a molar ratio of 3:2 and incubated for 15 min at room temperature. Reactions were then run on SDS-PAGE. STMBP and each SpyCatcher construct were also prepared individually as a reference for unreacted components. Lane 2: STMBP, Lane 3: SpyCatcher, Lane 4: SpyCatcher + STMBP, Lane 5: Penetratin-SpyCatcher, Lane 6: Penetratin-SpyCatcher + STMBP, Lane 7: CUPID-SpyCatcher, Lane 8: CUPID-SpyCatcher + STMBP, Lane 9: pVEC-SpyCatcher, Lane 10: pVEC-SpyCatcher + STMBP.

Figure 4.

Circular Dichroism Spectroscopy of SpyCatcher003 and CPP fusions. Monomeric SpyCatcher003 and CPP fusions were analyzed by CD. Proteins were mixed either with phosphate buffer or phosphate buffer containing 0.1% SDS.

Figure 5.

Lattice SIM imaging of stained bacteria shows internalization of SpyCatcher and CPP fusions. E. coli cells were treated with AF647 labeled CPP-SpyCatcher003 or SpyCatcher003 proteins. Cells were then washed and stained with FM4-64 to stain the outer membranes of cells. Cells were imaged using LatticeSIM to allow for subdiffraction localization of the labeled protein. Scale bar represents 5 μm.

Figure 6.

Lattice SIM imaging of mid-log phase bacteria show no internalization of SpyCatcher fusions. E. coli cells were treated with AF647 labeled CPP-SpyCatcher003 or SpyCatcher003 proteins. Cells were then washed and stained with FM4-64 to stain the outer membranes of cells. Cells were imaged using LatticeSIM to allow for subdiffraction localization of the labeled protein. Scale bar represents 2 μm in each image.

Figure 7.

Lattice SIM imaging of Caulobacter crescentus cells show Pentratin increases internalization of SpyCatcher. (A) Caulobacter crescentus cells were treated with AF647 labeled CPP-SpyCatcher003 or SpyCatcher003 proteins. Cells were then washed and stained with Nuc Green to stain the outer membranes of cells. Cells were imaged using LatticeSIM to allow for subdiffraction localization of the labeled protein. The scale bar represents 2 μm. (B) Quantitation of AF647 fluorescence inside cells. The mean fluorescence intensity for each construct was measured from dual color images as in (A) and is plotted as a violin plot. For CUPID-Spycatcher N = 582, for Pentratin-SpyCatcher N = 264, and for SpyCatcher N = 492.