Extracellular production of N-glycosylated Fn3 in Escherichia coli via optimized signal peptides, strains, and chemical reagents

Junwen Tan; Ning Ding; Xiaomei Zhang; Wei Li; Yue Lin; Qian Wang; Jiawei Li; Qiong Wu; Xuejun Hu; Ge Jiang; Yang Zheng

doi:10.1016/j.ijbiomac.2025.145778

Extracellular production of N-glycosylated Fn3 in Escherichia coli via optimized signal peptides, strains, and chemical reagents

Int J Biol Macromol. 2025 Jul 3:145778. doi: 10.1016/j.ijbiomac.2025.145778. Online ahead of print.

Authors

Junwen Tan¹, Ning Ding², Xiaomei Zhang², Wei Li³, Yue Lin³, Qian Wang³, Jiawei Li⁴, Qiong Wu², Xuejun Hu², Ge Jiang⁵, Yang Zheng⁶

Affiliations

¹ Medical College, Dalian University, Dalian, China; College of Life Health, Dalian University, Dalian, China.
² Medical College, Dalian University, Dalian, China; Dalian Key Laboratory of Oligosaccharide Recombination and Recombinant Protein Modification, Dalian, China; Dalian JR Binder LTD, Dalian, China.
³ Medical College, Dalian University, Dalian, China; Dalian Key Laboratory of Oligosaccharide Recombination and Recombinant Protein Modification, Dalian, China.
⁴ School of Software Technology, Dalian University of Technology, Dalian, China.
⁵ College of Life Health, Dalian University, Dalian, China.
⁶ Medical College, Dalian University, Dalian, China; Dalian Key Laboratory of Oligosaccharide Recombination and Recombinant Protein Modification, Dalian, China. Electronic address: zhengyang0713@foxmail.com.

PMID: 40617423
DOI: 10.1016/j.ijbiomac.2025.145778

Abstract

Fn3 3.4.4, an engineered variant of the fibronectin type III domain (Fn3), is a well-known extracellular matrix protein and regulator of tumorigenesis. The efficient excretion of Fn3 3.4.4 and its N-glycosylated form in Escherichia coli (E. coli) is crucial for its characterization and industrial production. In this study, we present a method that addresses this challenge by combining optimized signal peptides (SPs) and specific strains, along with the use of chemical reagents such as Triton X-100. We expressed glycosylated and aglycosylated Fn3 3.4.4, along with three different SPs-outer membrane protein A (OmpA), pectate lyase B (PelB), and L-asparaginase II (L-AsPsII)-in E. coli BL21 (DE3) or E. coli DKK601, followed by Triton X-100 treatment. We investigated the excretion of Fn3 3.4.4 and N-glycosylated Fn3 3.4.4 using these systems. PelB SP efficiently directed soluble Fn3 3.4.4 into the periplasm by virtue of its high cleavage rate. Additionally, N-glycosylated Fn3 3.4.4, led by OmpA SP, was detected in the cultivation medium while retaining its binding affinity to mesothelin (MSLN), as confirmed by Enzyme-linked immunosorbent assay (ELISA). These results suggest that these approaches could serve as a foundation for the characterization and large-scale production of N-glycosylated Fn3 3.4.4 in E. coli.

Keywords: Escherichia coli; Extracellular production; Fibronectin type III domain; N-glycosylation; Signal peptide.