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In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling

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Abstract

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.

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Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (No. 30771500), the Shanghai Science and Technology Committee, and the Shanghai Leading Academic Discipline Project (Project Number: B209).

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Authors and Affiliations

  1. Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, 200240, People’s Republic of China

    Xue-Fei Wang, Qi-Zhang Li, Ting-Wen Bao, Wei-Ran Cong & Xuan-Wei Zhou

  2. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA

    Wen-Xia Song

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  1. Xue-Fei Wang
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Correspondence to Xuan-Wei Zhou.

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Xue-Fei Wang and Qi-Zhang Li contributed equally to this work and share first authorship.

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Wang, XF., Li, QZ., Bao, TW. et al. In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling. Appl Microbiol Biotechnol 97, 2455–2465 (2013). https://doi.org/10.1007/s00253-012-4131-z

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