Screening of Muteins Secreted by Yeast: Random Mutagenesis of Human Interleukin-2

Abstract

We have generated random variants of human interleukin-2 (hIL-2) and assayed them using a rapid method that is generally applicable to proteins secreted by yeast. Mutein screening is performed in three steps: (1) in vitro mutagenesis of the target gene and selection of mutant genes by a physical method, (2) insertion of mutant genes into a yeast secretion vector, followed by yeast transformation, and (3) biological assay of protein variants secreted by the transformants into the culture fluid. In this way, we produced and tested about 500 variants of hIL-2 for activity; 155 variants were found to be biologically inactive. Sequence analyses of genes encoding the inactive variants revealed residues essential for hIL-2 activity, including residues at the extreme C-terminus and within a C-terminal α-helix (helix E).

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Ernst, J., Richman, L. Screening of Muteins Secreted by Yeast: Random Mutagenesis of Human Interleukin-2. Nat Biotechnol 7, 716–720 (1989). https://doi.org/10.1038/nbt0789-716

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