Abstract: Objective Human alpha-lactalbumin made lethal to tumor cells (HAMLET) is a novel potent tumoricidal agent that selectively induces apoptosis-like cell death in a variety of cancers.Methods As the sufficient amount and concentration of human alpha-lactalbumin (HLA) to form HAMLET is critical for its anti-tumor activity, mass production and rapid purification of recombinant HLA protein is highly desired. We first constructed a cDNA library of MCF-7 cell, known to secrete mature HLA peptides. Then amplified the full-length of HLA coding sequence by PCR, and cloned it into a pET30a(+) vector with an C-terminus His-tag. The His-rHLA was highly expressed in BL21 E coli cells with the induction of IPTG and purified by Ni-NTA affinity chromatography. The rHLA was refolded in an in vitro refolding buffer, de-calcinated with EDTA treatment, and then reacted with oleic acid (OA) water suspension under heated conditions to form the HAMLET complex. Results By comparing our HAMLET product with a reference preparation (BAMLET) from bovine alpha-lactalbumin and OA, we found that our preparations of HAMLET have a similar UV spectrum and a comparable tumoricidal activity as determined by cell viability assays and morphologic observations.Conclusion The cost-effectiveness and scalability of HAMLET production using recombinant HLA can be extremely useful for the further investigation of mechanistic studies of HAMLET functions, and may also be potentially considered as a practical anti-cancer agent for therapeutic trials.

Key words: human alpha-lactalbumin made lethal to tumor cells, alpha-lactalbumin, tumor cell, programmed cell death