Abstract
Because of the limitations of standard culture techniques, the development of new recombinant protein expression systems with biotechnological potential is a key challenge. Ideally, such systems should be able to effectively and accurately synthesize a protein of interest with intrinsic metabolic capacity. Here, we describe such a system that was designed based on a plasmid vector containing promoter elements derived from the metallothionein MTIIa promoter, as well as processing and purification elements. This promoter can be induced by heavy metals in a culture medium to induce the synthesis of human prostate-specific antigen (hPSA), which has been modified to insert elements for purification, proteolysis, and secretion. We optimized hPSA production in this system by comparing the effects and contributions of ZnCl2, CdCl2, and CuSO4 in HEK293FT, HeLa, BHK-21, and CHO-K1 cells. We also compared the effectiveness of three different transfection agents: multi-walled carbon nanotubes, Lipofectamine 2000, and X-tremeGENE HP Reagent. hPSA production was confirmed via the detection of enhanced green fluorescent protein fluorescence, and cell viability was determined. The expression of hPSA was compared with that of the native protein produced by LNCaP cells, using enzyme-linked immunosorbent assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis. X-tremeGENE reagent, the BHK-21 cell line, and CuSO4 showed the highest hPSA production rates. Furthermore, BHK-21 cells were more resistant to the oxidative stress caused by 100 μM CuSO4. These results suggest that the proposed optimized inducible expression system can effectively produce recombinant proteins with desired characteristics for a wide range of applications in molecular biology.
Highlights
The production of highly complex proteins is preferably performed in target expression eukaryotic cell cultures
The enhanced green fluorescent protein (eGFP) reporter was co-expressed with human prostate-specific antigen (hPSA) and its detection was used as a direct confirmation of the expression of hPSA protein measured by enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE
We developed an inducible expression system based on the use of a plasmid expression vector containing a reduced region of the MTIIa promoter that included the hPSA gene modified for high efficiency processing and purification
Summary
The production of highly complex proteins is preferably performed in target expression eukaryotic cell cultures. Insect cells transformed by baculovirus infection have gained popularity, owing to their high growth rate and the scale on which they can be produced. These cells are useful because of their resistance to metabolic stress. Other limitations related to baculovirus infection mechanisms and the incorrect folding and secretion of proteins limit their use for certain types of proteins (Treanor, 2009; Walsh, 2014). Metallothioneins were discovered in 1957 as cadmium protein ligands in equine kidneys. They are small, cysteine-rich proteins that play a fundamental role in the homeostasis of heavy metals. Transcription is regulated by these metals, but other stimuli such as hypoxia, oxidative stress, or hormones have been described (Kagi and Valee, 1960; Haslinger and Karin, 1985)
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