The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.
The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris.
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以荧光蛋白iLOV为报告基因筛选高产抗菌肽的毕赤酵母。
DOI：
10.1111/1751-7915.14034
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发表时间：
2022-07
影响因子：
5.7
通讯作者：
中科院分区：
文献类型：
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作者：
甲醇营养型酵母巴斯德毕赤酵母（Pichia pastoris）通常用于大规模生产重组蛋白。转化后最佳过表达菌株的鉴定可能是耗时和试剂消耗的。荧光报告基因如GFP已被用于协助鉴定上级生产者，但其相对较大的尺寸、成熟要求和狭窄的温度范围限制了其应用。在这里，我们介绍使用iLOV（一种基于黄素的荧光蛋白）作为荧光标记物，轻松快速地识别巴斯德毕赤酵母高产菌株。这种荧光蛋白作为融合伴侣的用途通过抗微生物肽NI01的产生来举例说明，NI01是一种难以以其天然形式过表达的靶标。iLOV荧光与蛋白质表达水平和染色体整合基因的拷贝数良好相关。在转化后直接进行简单的中通量平板筛选可实现低复杂性筛选，而使用荧光激活细胞分选（FACS）的高通量方法可实现全面的文库筛选。测试了iLOV_NI01融合盒的密码子优化和整合到巴斯德毕赤酵母基因组中的不同策略，以产生和分离高产菌株。通过iLOV报告基因的可视化和有效切割，检查遗传稳定性、工艺再现性和活性天然肽的纯化变得容易。我们表明，该系统可用于表达和筛选几种不同的抗菌肽重组产生的巴斯德毕赤酵母。甲醇营养型酵母巴斯德毕赤酵母通常用于大规模生产重组蛋白，但在转化后鉴定最佳过表达菌株可能耗时且耗费试剂。在这里，我们介绍了使用iLOV，一种基于黄素的荧光蛋白，作为一个可移动的荧光标记，以识别巴斯德毕赤酵母头奖菌株容易和快速。我们表明，该系统可用于表达，筛选和监测的遗传稳定性和工艺重现性的几种不同的抗菌肽重组产生的毕赤酵母。
The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin‐based fluorescent protein, as a fluorescent marker to identify P. pastoris high‐yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium‐throughput plate‐based screen directly following transformation is demonstrated for low complexity screening, while a high‐throughput method using fluorescence‐activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high‐yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris. The methylotrophic yeast Pichia pastoris is commonly used for production of recombinant proteins at scale, but identification of an optimally over‐expressing strain following transformation can be time and reagent consuming. Here we introduce the use of iLOV, a flavin based fluorescent protein, as a removable fluorescent marker to identify P. pastoris jackpot strains easily and rapidly. We show that this system can be used for expression, screening, and monitoring of genetic stability and process reproducibility of several different antimicrobial peptides recombinantly produced in Pichia.
通过增加遗传冗余提高非常规酵母的基因打靶效率
影响因子：
3.7
作者：
Chen Z;Sun H;Li P;He N;Zhu T;Li Y
通讯作者：
Li Y