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

Yasuo Mitani,Shusei Kanie,Nobutaka Mitsuda,Azusa Tomioka,Kwimi Chung,Yoshihiro Ohmiya,Hiroyuki Kaji,Rie Yasuno,Kiyohito Kihira

doi:10.3389/fbioe.2022.774786

Abstract

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.

Highlights

Luciferase is an enzyme that oxidizes the substrate luciferin; some are cellular proteins, while others are secreted
We reported the expression of recombinant CLuc deficient in N-glycosylation binding sites (Dmt CLucCO) in a mammalian adherent cell, COS1
We first focused on Expi293F cells, which have been adapted to grow in high density and to give higher recombinant protein yields, and tried a suspension culture of the Expi293 expression system, with the expectation of high expression of CLuc

Summary

Introduction

Luciferase is an enzyme that oxidizes the substrate luciferin; some are cellular proteins, while others are secreted. Secreted luciferases have been reported in a wide range of phyla including Arthropoda, Mollusca, and Annelida (Shimomura 2012). Luciferases from the genera Oplophorus and Gaussia have been widely used in application research (Hall et al, 2012; Suzuki and Inouye 2014; Markova et al, 2019). Oplophorus luciferase (OLuc) has some advantages, such as its catalytic domain with low molecular weight, excellent heat stability, and relatively easy expression in both prokaryotic and eukaryotic cells when some amino acid mutations are incorporated (Hall et al, 2012; Inouye et al, 2013). The three-dimensional (3D) structures of these luciferases have been elucidated by crystallization and X-ray diffraction of the partial domain exhibiting the catalytic function in OLuc (Tomabechi et al, 2016) and by heteronuclear NMR for GLuc (Wu et al, 2020)

Methods

Results

Conclusion