Optimization of Human Papillomavirus type 18 E6 recombinant oncoprotein expression in HEK293T cells

CONTRERAS NE; ROLDÁN JS; CASTILLO DS

Mendoza

Congreso; LVIII Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2022

Human papillomavirus (HPV) is a small, non enveloped double-stranded DNA virus. More than 150 HPV types have been identified, and at least 12 of these have been defined as cancer causing. HPV16 and HPV18 variants are together responsible for 70% of all cervical cancers. The E6 and E7 oncoproteins are responsible for the onset and maintenance of the cell transformation state by causing uncontrolled cell division and genome instability. They are the main targets for vaccine development and for diagnostic and therapeutic tools design. Several failed attempts have been made to produce recombinant full-length E6 protein in its native and soluble form in Escherichia coli. Given the difficulties found to express the HPV E6 protein in a prokaryotic host, we aimed at establishing an optimized recombinant HPV18 E6 mammalian expression system in order to purify this oncoprotein, which can potentially be used in the development of diagnostic tests.An increase in protein production in mammalian cells can be achieved by optimizing the culture medium or by the addition of small molecule enhancers (SME) to the media. HEK293T HPV18 E6 expressing cells were obtained by transient transfection with pcDNA3.1 (+) vector containing HPV18 E6 coding sequence fused to an N-terminal secretory signal peptide and a C-terminal 6xHis tag. First, we assessed whether modifications in the culture medium could have an effect on HPV18 E6 secretion in transfected HEK293T cells. Under serum depletion conditions, we observed an upregulation of HPV18 E6 protein levels in the supernatant of HEK293T cells, with a maximum at day 3 of incubation. Next, we tested whether addition of SME could further induce HPV18 E6 secretion to the supernatant in serum-starved HEK293T cells. Optimization of HPV18 E6 protein secretion was accomplished with 2 mM and 4 mM sodium butyrate treatment in serum-free media incubation for 3 days. Addition of rapamycin or hypotonic shock treatment did not enhance HPV18 E6 production in serum starved HEK293T cells. Cell viability in HPV18 E6 HEK293T expressing cells in the presence of sodium butyrate was evaluated by Alamarblue reduction assay. We observed that 2 mM sodium butyrate had a protective effect in HEK293T cells incubated in serum-starved media. Finally, HPV18 E6 oncoprotein was purified by nickel affinity chromatography from the supernatant of transiently transfected HEK293T cells exposed to 2 mM sodium butyrate and incubated in serum-free media for 3 days. The identity of the purified HPV18 E6 protein was confirmed by immunoblotting Additionally, we performed a cross-linking assay using different glutaraldehyde concentrations, which resulted in the formation of a predominant complex with a molecular mass compatible with a trimeric form.The obtained HPV18 E6 recombinant oncoprotein is a reliable biological tool that can potentially be applied in the development of diagnostic tests to prevent cervical cancer.