Within the field of protein-based biomaterials, the need exists for both covalent and oriented bioconjugation strategies for improved performance. Such bioconjugation reactions can be facilitated by engineering proteins with chemically activated amino acids at strategically chosen sites. The incorporation of these unnatural amino acids (uAAs) can be achieved by using the nonsense suppression technique. This requires an aminoacyl-tRNA-synthetase (aaRS) that exclusively recognizes the uAA and loads it to the corresponding tRNA. Appropriate (aaRS) mutants can be found through reverse engineering using the Saccharomyces cerevisiae strain MaV203. This strain contains a counterselectable, Gal4p-inducible SPAL10::URA3 fusion and deletions in the endogenous GAL80 and GAL4 genes. Therefore, it has been used extensively for the screening of aaRS mutant libraries. It is generally assumed that the SPAL10 promoter actively represses the URA3 gene in the absence of Gal4p, resulting in MaV203 cells with a Ura- phenotype. The current contribution reveals that in a small fraction of MaV203 cells, a basal expression of the URA3 gene occurs. The unexpected URA3 expression is reported for the first time, and the nature of the mutation causing this expression was identified as a spontaneous recessive mutation in a single gene of a protein involved in the repression of the SPAL10 promoter. The basal URA3 expression causes aaRS mutants to be missed, which affects the outcome of the library screening. It is demonstrated that the use of diploid cells can circumvent the MaV203 Ura+ phenotype, allowing for an optimization of S. cerevisiae library screening.

中文翻译：

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对MaV203酿酒酵母中URA3基因的自发性抑制的更深入了解及其对蛋白质工程和反向双杂交系统的影响。

在基于蛋白质的生物材料领域中，需要共价和定向的生物缀合策略以改善性能。通过在战略选择的位点用化学活化的氨基酸工程化蛋白质，可以促进这种生物偶联反应。这些非天然氨基酸（uAAs）的掺入可以通过使用废话抑制技术来实现。这需要专门识别uAA并将其加载到相应tRNA的氨酰基tRNA合成酶（aaRS）。适当的（aaRS）突变体可以使用酿酒酵母菌株MaV203通过逆向工程找到。该菌株在内源性GAL80和GAL4基因中包含一个可逆选择的，Gal4p诱导的SPAL10 :: URA3融合和缺失。因此，它已被广泛用于筛选aaRS突变体文库。通常认为SPAL10启动子在不存在Gal4p的情况下会主动抑制URA3基因，导致MaV203细胞具有超表型。目前的贡献表明，在MaV203细胞的一小部分中，发生了URA3基因的基础表达。首次报道了意外的URA3表达，并且导致该表达的突变的性质被确定为参与SPAL10启动子阻遏的蛋白质单个基因中的自发性隐性突变。URA3的基础表达导致aaRS突变体丢失，从而影响文库筛选的结果。已证明使用二倍体细胞可以规避MaV203 Ura +表型，从而优化S。