A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans.
A new insect cell line engineered to produce recombinant glycoproteins with cleavable N-glycans.
复制标题
DOI：
10.1016/j.jbc.2021.101454
复制
发表时间：
2022-01
期刊：
影响因子：
--
通讯作者：
Jarvis DL
中科院分区：
文献类型：
--
作者：
Mabashi-Asazuma H;Jarvis DL
糖蛋白很难结晶，因为它们具有由多个单糖组成的多相聚糖，它们的o -糖苷键具有相当大的旋转自由。研究n -糖蛋白的晶体学家通常通过使用缺乏β1,2- n -乙酰氨基葡萄糖转移酶I （MGAT1）的哺乳动物细胞系来规避这个问题，这些细胞系产生含有未成熟n -聚糖的重组糖蛋白。这些聚糖支持蛋白质折叠和质量控制，但可以使用内do-β- n -乙酰氨基葡萄糖酶H （endo H）去除。许多晶体学家也使用杆状病毒-昆虫细胞系统（BICS）来生产重组蛋白，但没有途径获得缺乏mgat1的昆虫细胞系来促进该系统中的糖蛋白结晶。因此，我们使用bbics特异性CRISPR-Cas9载体编辑了横纹病毒阴性Spodoptera frugiperda细胞系（Sf-RVN）的Mgat1基因，并分离出一个具有多个Mgat1缺失的亚克隆，我们将其命名为Sf-RVNLec1。我们发现Sf-RVN和Sf-RVNLec1细胞具有相同的生长特性，并且同样可以作为杆状病毒介导的重组糖蛋白生产的宿主。n -聚糖分析显示，从Sf-RVNLec1细胞中分离的总内源性糖蛋白片段只有未成熟的高甘露糖型n -聚糖。最后，n -聚糖谱分析和内糖苷酶分析表明，Sf-RVNLec1细胞产生的三种重组糖蛋白上的n -聚糖绝大多数是Endo h -可切割的Man5GlcNAc2结构。因此，本研究为BICS提供了一种新的昆虫细胞系，可用于生产具有Endo h -可切割n -聚糖的重组糖蛋白。这将使研究人员能够将BICS的高生产率与重组糖蛋白去糖基化的能力结合起来，这将有助于通过x射线晶体学确定糖蛋白结构。
Glycoproteins are difficult to crystallize because they have heterogeneous glycans composed of multiple monosaccharides with considerable rotational freedom about their O-glycosidic linkages. Crystallographers studying N-glycoproteins often circumvent this problem by using β1,2-N-acetylglucosaminyltransferase I (MGAT1)–deficient mammalian cell lines, which produce recombinant glycoproteins with immature N-glycans. These glycans support protein folding and quality control but can be removed using endo-β-N-acetylglucosaminidase H (Endo H). Many crystallographers also use the baculovirus-insect cell system (BICS) to produce recombinant proteins for their work but have no access to an MGAT1-deficient insect cell line to facilitate glycoprotein crystallization in this system. Thus, we used BICS-specific CRISPR–Cas9 vectors to edit the Mgat1 gene of a rhabdovirus-negative Spodoptera frugiperda cell line (Sf-RVN) and isolated a subclone with multiple Mgat1 deletions, which we named Sf-RVNLec1. We found that Sf-RVN and Sf-RVNLec1 cells had identical growth properties and served equally well as hosts for baculovirus-mediated recombinant glycoprotein production. N-glycan profiling showed that a total endogenous glycoprotein fraction isolated from Sf-RVNLec1 cells had only immature and high mannose-type N-glycans. Finally, N-glycan profiling and endoglycosidase analyses showed that the vast majority of the N-glycans on three recombinant glycoproteins produced by Sf-RVNLec1 cells were Endo H-cleavable Man5GlcNAc2 structures. Thus, this study yielded a new insect cell line for the BICS that can be used to produce recombinant glycoproteins with Endo H-cleavable N-glycans. This will enable researchers to combine the high productivity of the BICS with the ability to deglycosylate recombinant glycoproteins, which will facilitate efforts to determine glycoprotein structures by X-ray crystallography.
登录
查看更多内容
影响因子：
7.7
作者：
Drinnenberg IA;deYoung D;Henikoff S;Malik HS
通讯作者：
Malik HS
影响因子：
3.5
作者：
Geisler C
通讯作者：
Geisler C
影响因子：
4.3
作者：
ALTMANN, F;KORNFELD, G;GLOSSL, J
通讯作者：
GLOSSL, J
影响因子：
5.6
作者：
Gordon, Roni D.;Sivarajah, Prashanth;Rini, James M.
通讯作者：
Rini, James M.
影响因子：
6.8
作者：
Columbus, Linda
通讯作者：
Columbus, Linda