Nuruliawaty Utami, Dini Nurdiani, Hariyatun Hariyatun, Eko Wahyu Putro, Fadillah Putri Patria, Wien Kusharyoto
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This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM).</p><p><strong>Results: </strong>ImageJ analysis of the HIP's SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones.</p><p><strong>Conclusions: </strong>CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. 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引用次数: 0
摘要
背景:人胰岛素是美国食品和药物管理局批准的第一种通过 DNA 重组技术生产的生物制药药物。之前的研究成功地在 Pichia pastoris 中表达了重组人胰岛素前体(HIP)的截短和全长 α-因子重组克隆。Matting α-因子(Matα)是一种信号分泌物,可引导 HIP 蛋白进入培养基。本研究旨在比较在两种培养基(缓冲甲醇复合培养基(BMMY)和甲醇基础盐培养基(BSMM))中生长的全长和截短α-因子分泌信号克隆的HIP表达情况:ImageJ对HIP的SDS-PAGE分析表明,重组牧马人截短α-因子克隆(CL4)在两种培养基中表达的HIP平均表达水平明显高于全长(HF7)。Western 印迹分析表明,表达的蛋白是 HIP。使用 AlphaFold 预测了α-因子蛋白的结构,并使用 UCSF ChimeraX 进行了可视化,以确认两个克隆的分泌能力:结论:CL4克隆在P. pastoris HIP表达盒中使用了截短的α-因子,其HIP表达量是使用全长α-因子分泌信号的HF7克隆的8.97倍(在BMMY中)和1.17倍(在BSMM中)。这项研究证实,删除分泌信号序列的某些区域可显著提高牧杆菌中 HIP 蛋白的表达效率。
Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison.
Background: Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM).
Results: ImageJ analysis of the HIP's SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones.
Conclusions: CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.