Improving recombinant protein expression is a perpetual goal for molecular pharming. However, over-transcription of recombinant protein induces ER stress, and causes protein degradation. Here, we describe a knock-in approach to integrate a human serum albumin expression cassette into the locus of the rice storage protein GluA1 by site-specific integration via the nonhomologous end joining (NHEJ) pathway. The expression level of OsrHSA in the knock-in (KI) lines was much higher than that of the random integration (RI) lines. ER stress in KI line endosperm cells was not significantly altered even after massive OsrHSA accumulation in rice endosperm cell. Instead, ER stress induced by high OsrHSA expression was alleviated in the KI line via the inositol-requiring enzyme 1 (IRE1)-mediated/OsbZIP50 pathway. Furthermore, improvement of OsrHSA expression in KI lines is likely due to reduction of contents of glutelin and globulin in rice endosperm cell. These results provide insight into an approach to improving recombinant protein accumulation by alleviating ER stress and protein trafficking.

改善重组蛋白表达是分子修饰的永恒目标。但是，重组蛋白的过度转录会引起内质网应激，并导致蛋白降解。在这里，我们描述了一种将人血清白蛋白表达盒整合到水稻贮藏蛋白GluA1基因座中的敲入方法通过非同源末端连接（NHEJ）途径进行位点特异性整合。OsrHSA在敲入（KI）系中的表达水平远高于随机整合（RI）系。即使水稻胚乳细胞中大量OsrHSA积累，KI系胚乳细胞中的ER胁迫也没有显着改变。相反，KIs系中通过需要肌醇的酶1（IRE1）介导的/ OsbZIP50途径缓解了由高OsrHSA表达诱导的ER应激。此外，可能由于稻胚乳细胞中谷蛋白和球蛋白含量的减少而改善了KI系中OsrHSA表达。这些结果提供了通过减轻内质网应激和蛋白质运输来改善重组蛋白质积累的方法的见识。