Low-scale production and purification of a biologically active optimized form of the antitumor protein growth arrest specific 1 (GAS1) in a mammalian system for post-translational analysis

Abstract

Growth arrest specific 1 (GAS1) is a protein involved in cell arrest that induces quiescence and apoptosis, when it is overexpressed in tumor cells. We have previously reported the generation of a soluble truncated protein known as tGAS1, with antitumoral capacity. In this paper two lentiviral constructs were developed, containing: the native human gene sequence fused to a Hisx6-tag (tG1Hc); or a synthetic and optimized DNA sequence (tG1Hs). Both transgenes encode for a version of the human tGAS1 protein with the addition of a Hisx6-tag in the C-terminus (tG1H). Resultant VSV-G pseudotyped lentiviruses were used to generate HEK-293FT and U87-MG cell cultures stable for the constitutive production of tG1H proteins. The data show that only U87-MG expressed the recombinant proteins when infected. In fact, conditioned medium obtained from U87-MG cells producing tG1Hs mRNA preserves anti-proliferative and pro-apoptotic properties. Noteworthy, viral particles carrying the tG1Hs transgene inhibit tumor growth in a heterotopic xenograft mice model. Additionally, the recombinant tG1H protein was purified by multiple affinity protocols and used to perform further experiments to analyze post-translational processing or affinity co-precipitation protocols. In the present work we have studied the N-glycosylation of GAS1 in the purified protein.