AIMS Press Acta Medica Okayama 2377-9098 7 2 2020 The expression level and cytotoxicity of green fluorescent protein are modulated by an additional N-terminal sequence 121 132 EN Hisao Moriya Graduate School of Environmental and Life Sciences, Research Core for Interdisciplinary Sciences, Okayama University Nucleotide and amino acid sequences at the N-terminus affect the expression level and cytotoxicity of proteins; however, their effects are not fully understood yet. Here, N-terminal 30 nucleotide/10 amino acid (N10) sequences that affect the expression level and cytotoxicity of a green fluorescent protein were systematically isolated in the budding yeast Saccharomyces cerevisiae. The expression per gene (EPG) and gene copy number limit (CNL) relationships were examined to assess the effects of the N10 sequence. The isolated N10 nucleotide sequences suggested that codon optimality is the major determinant of the protein expression level. A higher number of hydrophobic or cysteine residues in the N10 sequence seemed to increase the cytotoxicity of the protein. Therefore, a high frequency of specific amino acid residues in the outside of the main tertiary structure of proteins might not be preferable. No potential conflict of interest relevant to this article was reported.