Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vecto...Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vector p APtag at the N terminus of alkaline phosphatase(AP)for fusion expression.Next,293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.Then,by adding N-glycosylation processing inhibitor kifunensine into the medium,complex glycan was inhibited to generate.The residual glycan of purified protein was removed by endoglycosidase H.Finally,AP protein was removed by using human rhinovirus protease and size exclusion chromatography.The concentration of purified Necl-4 protein was monitored by measuring the absorbance at280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector.The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system,we can easily screen the high productive stable cell lines by using AP activity assay.By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H,we can obtain deglycosylated Necl-4 protein in milligram quantities.Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.展开更多
基金Supported by grants of the National Natural Science Foundation of China(31071203,31370789,31670789,and 31671316)
文摘Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vector p APtag at the N terminus of alkaline phosphatase(AP)for fusion expression.Next,293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.Then,by adding N-glycosylation processing inhibitor kifunensine into the medium,complex glycan was inhibited to generate.The residual glycan of purified protein was removed by endoglycosidase H.Finally,AP protein was removed by using human rhinovirus protease and size exclusion chromatography.The concentration of purified Necl-4 protein was monitored by measuring the absorbance at280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector.The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system,we can easily screen the high productive stable cell lines by using AP activity assay.By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H,we can obtain deglycosylated Necl-4 protein in milligram quantities.Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.
