Studies with Site-Specific Labeled HAMLET Figure 10. Reduced size HAMLET has comparable properties to complex derived from full-length protein. aD-hLA in complex with OA has biological properties that are similar to mg-hLA OA complex with cancer cell lines SK-BR-3 and MDA-MD-468 (data not shown). aD-hLA and aD- hLA-ext, complexed with OA have comparable tumoricidal activities measured by Trypan Blue (A) and by FACS analysis using Annexin V (B), indicating that the b-domain of hLA is not required for the complex to acquire cytotoxic properties. SK-BR-3 cells were incubated with Alexa Fluor 488 labeled aD-hLA-ext either alone (C) or as OA complex (D) for 3 h. The cells were then fixed with 4% PFA in PBS, and the nuclei were counterstained with Hoechst 33342 (blue). Only aD-hLA-ext-Alexa Fluor 488 (green signal) complexed with OA is internalized by the cells. doi:10.1371/journal.pone.0026093.g010 conjugation of a bioactive agent with an aminooxy group. In the present study, extending the a-LA with C-terminal fusion peptide was not expected to alter its biological activity, since a-LA from rat naturally exists with an 11–amino acid C-terminal extension [23]. The labeling was carried out before the OA complex was formed. We have observed that the native hLA with the N-terminal his-tag readily binds to a metal affinity column; however, after the OA complex is made, the protein does not bind to the metal affinity column. Therefore, the C-terminal labeling was carried out prior to making the OA complex. Furthermore, the C-terminal extension of the native a-LA by itself does not affect its activity either as lactose synthase (data not shown) or, as shown in the present study, as a HAMLET, indicating that the protein with the C-terminal extension carrying the bulky fluoroprobe can still be converted into tumoricidal complex. Fluoroprobe-labeled and complexed molten globule protein was used for live-/fixed-cell imaging. Alexa Fluor 488 signal could be detected at the cell surface and, with time, accumulating at the cell nucleus, as shown by Hakansson et al. [33]. Moreover, we have identified a subnuclear structure in which the green fluorescent signal of Alexa Fluor 488 accumulated in nucleoli, using anti- fibrillarin antibody. The significance of co-localization of a labeled complex with cell nucleoli is unknown. However, a variety of molecules that apparently have no role in ribosome assembly, have been found at nucleoli [39]. In addition, one study has suggested that unfolded proteins are stored in the nucleolus during stress [40]. In addition to using site-specific labeling for microscopy studies, we have shown that the site-specific biotin coupling could also be achieved, suggesting a potential application for ultra-structural studies, such as transmission electron microscopy. Biotinylated protein could be used to ‘‘fish-out’’ cancer cell surface ligands interacting with HAMLET. These specific interactions between the cell surface ligand(s) and HAMLET may be enough to initiate the apoptotic process(es) leading to tumor cell death. In conclusion, we have been successful in adding a chemical handle to HAMLET without affecting its biological activity, allowing us to trace the complex at the cell surface or inside the cell. The addition of a site-specific handle to a tumoricidal complex could be further exploited for isolation of partner/target molecules, providing insight into the tumor specificity of the complex. Materials and Methods Cloning, expression, and refolding of hLA, hLA-ext, T-hLA, T-hLA-ext, and His-hLA The human a-LA gene was cloned from a mammary gland cDNA library (Clontech, Mountain View, CA) into Nde I and PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26093