Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

TL;DR

This paper describes the creation of a plasmid that produces a green fluorescent protein-tagged version of cathepsin L and its effects in colorectal cancer cells.

Contribution

The novel contribution is the construction of a plasmid encoding a fluorescently tagged human cathepsin L for studying its expression and function in cancer cells.

Findings

A plasmid coding for human cathepsin L fused with green fluorescent protein was successfully constructed and transfected into HCT116 cells.

Expression of the tagged cathepsin L affected the proliferation and metabolic state of HCT116 cells within 24 hours post-transfection.

Endogenous cathepsin L levels were higher than the engineered fluorescent protein chimera in transfected cells.

Abstract

The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015) [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP) in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

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