Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris

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

“…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.…”

“…The expression vectors pPIC9K and pPIC9K‐ XynCDBFV carrying xylanase gene XynCDBFV (GenBank accession number: KP691331.1) expression cassettes were maintained in our laboratory (Huang et al., 2021 ; Lu et al., 2023 ). The SAG1 gene from E. tenella (GenBank accession number: AJ586531.2) was optimized to enhance expression, utilizing the codon usage bias of P. pastoris as referenced from the codon usage database ( http://www.kazusa.or.jp/codon/ ).…”

“…Induced expression was conducted in plastic test tubes and shake flasks following the method described previously (Lu et al., 2023 ). Approximately twelve individual transformant colonies were selected at random from MD plates and inoculated into plastic tubes containing 3 mL of BMGY medium, followed by incubation with shaking at 30°C and 220 rpm.…”

“…Fed‐batch fermentation of P. pastoris was conducted in a 15 L fermenter (EastBio Co. Ltd., Jiangsu, China), following the method previously described (Lu et al., 2023 ). Initially, a positive transformant was cultured in 50 mL YPD at 30°C and 220 rpm for 48 h. Subsequently, this culture was expanded to 200 mL YPD with a 1% inoculation until an OD of 6.0 was achieved.…”

“…At present, many recombinant xylanases have been stably and efficiently expressed (Fang et al., 2014 ). Moreover, xylanase has become an effective biotechnological tool as a fusion partner to enhance the expression level of proteins in P. pastoris recently (Lu et al., 2023 ).…”