Department of Biological Sciences, University of the Pacific Stockton, CA 95211, USA.
Gene. 2013 May 1;519(2):311-7. doi: 10.1016/j.gene.2013.01.062. Epub 2013 Feb 21.
The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.
甲醇营养酵母毕赤酵母已被基因工程改造，用于生产许多用于工业和研究目的的异源蛋白。为了分泌蛋白质，以便更易于从细胞外介质中纯化，重组蛋白的编码序列最初与酿酒酵母α-交配因子分泌信号序列融合。为了确定各种缺失和取代对表达的影响，对α-交配因子分泌信号序列的前导肽区进行了广泛的定点诱变。虽然一些突变明显降低了蛋白质的表达，但删除对应于α-交配因子分泌信号预测的第 3 个α螺旋的氨基酸 57-70，至少将报告蛋白辣根过氧化物酶和脂肪酶的分泌增加了 50%在小规模培养中。这些发现表明，该分泌信号的分泌效率在未来可能会进一步提高。
