Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line / 中国医学科学杂志(英文版)

Objective To screen the transient and stable cell lines with high production of Nectin-like 4 (Necl-4) protein. Methods First, cDNA sequences encoding the extracellular domain of Necls were cloned into the modified vector pAPtag 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 at 280 nm and analyzed by SDS-PAGE. Result 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.

Advances in CRISPR/Cas9 gene-editing system / 基础医学与临床

CRISPR/Cas9 gene-editing system has been broadly used in various fields of bioscience and medicine in recent years. The system can be guided by RNA to specific DNA site thus achieving targeted gene editing.Off-target effect and editing efficiency remain to be two crucial challeges to the system. Currently, a number of researches have been focused on the optimization of the system by reducing off-target effects and increasing editing efficiency, which may enhance its safety and expand its application.