Evaluating the use of Apo-neocarzinostatin as a cell penetrating protein

Abstract

Protein-ligand complex neocarzinostatin (NCS) is a small, thermostable protein-ligand complex that is able to deliver its ligand cargo into live mammalian cells where it induces DNA damage. Apo-NCS is able to functionally display complementarity determining regions loops, and has been hypothesised to act as a cell-penetrating protein, which would make it an ideal scaffold for cell targeting, and subsequent intracellular delivery of small-molecule drugs. In order to evaluate apo-NCS as a cell penetrating protein, we have evaluated the efficiency of its internalisation into live HeLa cells using matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry and fluorescence microscopy. Following incubation of cells with apo-NCS, we observed no evidence of internalisation.

Fig. 1.

CD spectra of unlabelled NCS(WT) and labelled NCS constructs.

Fig. 2.

Thermal denaturation curves of unlabelled NCS(WT) and labelled NCS constructs, monitored by CD.

Fig. 3.

MALDI-TOF mass spectrum of 1 : 1 ¹⁴N-NCS(+C)-biotin and ¹⁵N-NCS(+C)-biotin (10 pmol of each protein were mixed, then pulled down using streptavidin-coated magnetic beads).

Fig. 4.

MALDI-TOF mass spectrum of internalised NCS(+C)-biotin. [Cells were incubated with ¹⁴N-NCS(+C)-biotin (10 μM, 1 h at 37°C), then pronase (0.5 mg/ml, 200 μl). 15N-NCS(+C)-biotin (10 pmol) was added before lysis and pulldown of biotinylated species.]

Fig. 5.

MALDI-TOF mass spectrum of cell-associated NCS(+C)-biotin. [Cells were incubated with ¹⁴N-NCS(+C)-biotin (10 µM, 1 h, 37°C). ¹⁵N-NCS(+C)-biotin was added before lysis and pulldown of biotinylated species.]

Fig. 6.

Microscopy of HeLa cells treated with tetramethylrhodamine-labelled NCS (scale bars are 5 µm).