Recombinant expression of hen egg white lysozyme with the assistance of xylanase fusion partner inPichia pastoris

Abstract

Due to its bacteriolytic activity, hen egg white lysozyme (HEWL) is widely used in the feed, food, and pharmaceutical industries. However, its application is hindered by low protein expression levels in microbial expression systems. In this work, a novel fusion protein expression strategy was proposed for increasing the expression level of HEWL. First, HEWL, fused with a highly expressed fusion protein partner xylanase XynCDBFV, is expressed in Pichia pastoris . Secondly, a linker including endogenous protease cleavage sites was introduced between two fusion proteins in order to separate them directly during the secretion process. Finally, the results show that the supernatant of XynCDBFV-HEWL has a higher HEWL expression level and activity compared with HEWL only. It should be noted that the expression of HEWL reaches to about 3.5 g/L, and the activity of HEWL against Micrococcus lysodeikticus reaches to 1.50 × 10 5 U/mL in a fed-batch fermentation, which is currently the highest level of recombinant expression of an egg white-derived lysozyme. Taken together, we acquired bioactive HEWL for large-scale recombinant production in Pichia pastor is using a novel fusion protein expression strategy, which could then be used for a variety of applications.

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken

“…As fusion with xylanase XynCDBFV improves the protein expression of hen egg white lysozyme (HEWL), somatostatin and IGF‐1/LR3 IGF‐1, and the XynCDBFV‐somatostatin displays excellent antigenicity (Cui et al., 2022 ; Huang et al., 2021 ), we investigated whether xylanase could similarly enhance the expression level and antigenicity of SAG1. To test the hypothesis, the code optimized and synthesized SAG1 and XynCDBFV‐SAG1 gene sequences were inserted into the P. pastoris expression vector pPIC9K with the α‐factor leader sequence for recombinant proteins secretion.…”

“…XynCDBFV is a thermostable alkaline xylanase derived from a rumen fungus Neocallimastix patriciarum and highly recombinant expressed in P. pastoris (Fang et al., 2014 ; Han et al., 2017 ). Due to the high expression of XynCDBFV in P. pastoris , we have successfully fused it to several difficult‐to‐express proteins, such as somatostatin, HEWL and IGF‐1/LR3 IGF‐1, and these fusions greatly increased their expression levels in P. pastoris , while preserving their bioactivity (Cui et al., 2022 ; Huang et al., 2021 ; Lu et al., 2023 ). As shown in this study, fusion XynCDBFV with SAG1 did not increase the expression level of SAG1, but we found surprisingly that the fusion with XynCDBFV improved the immune protection of SAG1 against E. tenella , which is the first report that the fusion of xylanase with antigen improves the immune protection of antigen.…”

Preparation, Biological Activities, and Potential Applications of Hen Egg-Derived Peptides: A Review

“…At present, some studies have reported that some hen egg proteins can be successfully expressed using genetic engineering strategies. For instance, with the assistance of a xylanase fusion partner, hen egg-white lysozymes were recombinantly expressed in Pichia pastoris [ 71 ]. Therefore, a new research trend in the preparation of HEPs is genetic engineering strategies.…”