Soluble Expression Of GMYL6 And Promoting The Cytotoxicity Of Human NK Cells

Objectives 1 Identify and obtain the amino-acid sequence of gMYL6.2 To improve the solubility of gMYL6 expressed in E.coli,different polycationic amino acid tags composed of multiple arginines or lysines were fused at its C-terminal.3 To obtain the effect of gMYL6 and recombinant gMYL6 on NK cell function by cytotoxicity test.Methods 1 The peptide sequence was acquired through the EASYnLCII-Nano-HPLC system and software Xcalibur 2.0.7,and the biological synthesis information of gMYL6 was obtained by NCBInr database searching and BLAST analysis.2The heterogenous expression of gMYL6 has been constructed by introducing polycationic amino acid tag(1~10-arginine or 1~10-lysine)to its terminal with E.coli BL21(DE3)as host and pET21a(+)as the expression vector.The recombinant protein was evaluated by SDS-PAGE and quantitative analysis.At the same time,the effect of temperature on gMYL6 or gMYL6-6K expression was checked via setting different induction temperature.3 The effects of gMYL6 and gMYL6-6K on cytotoxicity and phenotype of cord blood-derived NK cells were detected by CCK8 assay and flow cytometry.Results 1 The amino-acid sequence of gMYL6 was obtained by 2D-Nano-LC-ESI-LTQ-Orbitrap MS/MS and BLAST analysis.2gMYL6 and its recombinant protein were obtained successfully by soluble expression strategy according to its biosynthesis information.The results of SDS-PAGE and quantitative analysis showed that the levels of soluble protein upon the addition of 1R,9R,6K,9K or 10 K to the C-terminus of gMYL6 were higher,and the highest level was 23.7425 mg/L.According to the results of inducing expression under different temperature(18?and 30?),gMYL6 and gMYL6-6K had slightly lower production at higher induced temperature.3 The results of CCK8 and flow cytometry showed that gMYL6 and gMYL6-6K could increase cytotoxicity of NK cells,percentage of CD16~+CD56~+cells and frequency of CD107a~+NK cells when the drug concentration was 25?l.Conclusions 1 The related fragment information of gMYL6 can be obtained quickly and efficiently by tandem mass spectrometry.2 The soluble expression of gMYL6 can be improved by adding polycation amino acid tag.3 This study discovered that gMYL6 and gMYL6-6K possess the capacity of promoting the NK cells' cytotoxicity.