Abstract
Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K6-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K6-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD600 under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn2+, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in E. coli cells.
Highlights
Bacterial surface display has been widely applied in scientific research and the science industry [1]
Two signal peptides and three charged polypeptides were anchored to the N-terminus of InaK-N (Fig 1A), generating the engineered Inak-Ns, which were termed as InaK-N (585bp), ssMalE-InaK-N (663bp), ssTorA-InaK-N (687bp), D6-InaK-N (603bp), E6-InaK-N (603bp), and K6-InaK-N (603bp) (Fig 1D)
Human ARG1, which is a trimer in its active form, was not displayed on the cell surface using current bacterial surface display systems [18]
Summary
Bacterial surface display has been widely applied in scientific research and the science industry [1]. Recombinant vaccines, and catalytic enzymes could be displayed on bacterial surfaces through appropriate surface anchors, which facilitated their further engineering and applications for certain purposes [2]. In gram-negative bacteria, two major secretion systems, the general secretion (Sec) and twin arginine translocation (Tat) pathways [3,4], have been identified to transport proteins to the extracellular milieu under the guidance of various N-terminal signal peptides. Most surface carriers in bacteria are surface presenting. Surface Immobilization of Human Arginase-1 in E. coli funding. 3. LI Yi, National Natural Science Foundation of China (CN). Authors: Zhen Zhang, Rongxin Tang, Lu Bian, and Meng Mei received the funding
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