Wheat-embryo cell-free protein expression system allows efficient production of a wide variety of proteins. Homogeneity of the end products is an important characteristic of an advanced cell-free system that will be used in a field of protein science such as structural biology. A translation enhancer such as the omega sequence derived from tobacco mosaic virus, that allows cap-independent translation of the mRNA in the cell-free system, is required for low-cost preparation of template mRNAs in the cell-free translation system. However, the use of translational enhancers often leads to unexpected byproducts. Several AUU codons in the omega sequence can potentially function as translation initiators. We confirmed that the in-frame AUU in the omega sequence functions as a non-canonical start codon and results in the extension of the N-terminus of the target protein in some cases. Investigation of the selectivity of non-canonical initiation codon under the control of omega sequence in the wheat-embryo cell-free system revealed that seven non-AUG codons, CUG, AUA, AUU, GUG, ACG, AUC, and UUG, are recognized as translation initiators. We found that the introduction of an in-frame stop codon just upstream of the target open reading frame is an efficient way to avoid unexpected byproducts. This minor but effective modification facilitates production of homogeneous proteins within the wheat-embryo cell-free protein expression system at the preparative scale. (C) 2010 Elsevier Inc. All rights reserved.