DataSheet1_Host-Dependent Producibility of Recombinant Cypridina noctiluca Luciferase With Glycosylation Defects.PDF

Cypridina noctiluca luciferase (CLuc) is a secreted luminescent protein that reacts with its substrate (Cypridina luciferin) to emit light. CLuc is known to be a thermostable protein and has been used for various research applications, including in vivo imaging and high-throughput reporter assays. Previously, we produced a large amount of recombinant CLuc for crystallographic analysis. However, this recombinant protein did not crystallize, probably due to heterogeneous N-glycan modifications. In this study, we produced recombinant CLuc without glycan modifications by introducing mutations at the N-glycan modification residues using mammalian Expi293F cells, silkworms, and tobacco Bright Yellow-2 cells. Interestingly, recombinant CLuc production depended heavily on the expression hosts. Among these selected hosts, we found that Expi293F cells efficiently produced the recombinant mutant CLuc without significant effects on its luciferase activity. We confirmed the lack of N-glycan modifications for this mutant protein by mass spectrometry analysis but found slight O-glycan modifications that we estimated were about 2% of the ion chromatogram peak area for the detected peptide fragments. Moreover, by using CLuc deletion mutants during the investigation of O-glycan modifications, we identified amino acid residues important to the luciferase activity of CLuc. Our results provide invaluable information related to CLuc function and pave the way for its crystallographic analysis.

海萤荧光素酶（Cypridina noctiluca luciferase，CLuc）是一种分泌型发光蛋白，可与其底物海萤荧光素（Cypridina luciferin）结合并发光。CLuc属于热稳定蛋白，已被广泛应用于活体成像、高通量报告基因检测等诸多研究领域。此前本团队为开展晶体结构分析，制备了大量重组CLuc，但该重组蛋白未能结晶，推测原因是其N-糖基化修饰存在异质性。本研究中，我们通过在N-糖基化修饰位点引入突变，分别利用哺乳动物Expi293F细胞、家蚕以及烟草BY-2细胞（tobacco Bright Yellow-2 cells），制备了无糖基化修饰的重组CLuc。值得注意的是，重组CLuc的表达产量高度依赖宿主表达体系。在上述筛选的宿主体系中，我们发现Expi293F细胞可高效表达重组突变型CLuc，且对其荧光素酶活性无显著影响。本研究通过质谱分析证实该突变蛋白不存在N-糖基化修饰，但检测到微量O-糖基化修饰，经估算其占检测肽段的离子色谱峰面积的约2%。此外，在探究O-糖基化修饰的过程中，我们通过构建CLuc缺失突变体，鉴定出了对CLuc荧光素酶活性至关重要的氨基酸残基。本研究结果为CLuc功能相关研究提供了宝贵信息，同时为其晶体结构分析铺平了道路。