The human chromogranin A-derived peptide CGA-N12, which is composed of 12 amino acid residues with the sequence ALQGAKERAHQQ, showed strong antifungal activity and the least hemolytic activity in previous studies. However, synthetic peptides are relatively expensive to produce. Recombinant expression of peptides in the host cells, such as bacteria or yeast, can fastly provide cost-efficient products of peptides. Here, we developed an innovative system to produce CGA-N12 peptides in the yeast Pichia pastoris GS115 using genetic engineering technology. In order to directly secret short CGA-N12 peptides into the culture media from GS115 cells and enhance its expression effect, the structure of the CGA-N12 coding sequence was designed to mimic that of native α-factor gene of Saccharomyces cerevisiae. Four long primer pairs with sticky end were used to synthesize CGA-N12 expression sequence which contains four copies of CGA-N12 flanked by a Lys-Arg pair and two Glu-Ala repeating units. Endogenous proteases Kex2 and Ste13 in Golgi apparatus recognize and excise Lys-Arg and Glu-Ala pair to release short CGA-N12 peptides from the tandem repeat sequences, respectively. The CGA-N12 peptides were successfully expressed in Pichia pastoris with a yield of up to 30 mg/L of yeast culture as determined using HPLC. Our study indicated that the strategy employed in this work may be a good way to express small-molecule peptides directly in the Pichia pastoris system.

中文翻译：

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嗜铬粒蛋白A衍生的抗真菌肽CGA-N12在巴斯德毕赤酵母中的表达。

人嗜铬粒蛋白A衍生肽CGA-N12由12个氨基酸残基组成，序列为ALQGAKERAHQQ，在以前的研究中显示出很强的抗真菌活性和最小的溶血活性。但是，合成肽的生产相对昂贵。肽在宿主细胞（例如细菌或酵母菌）中的重组表达可快速提供具有成本效益的肽产品。在这里，我们开发了一种创新的系统，可使用基因工程技术在酵母毕赤酵母GS115中生产CGA-N12肽。为了将短的CGA-N12肽直接从GS115细胞分泌到培养基中并增强其表达效果，设计了CGA-N12编码序列的结构以模仿酿酒酵母的天然α因子基因。使用具有粘性末端的四个长引物对来合成CGA-N12表达序列，该序列包含四个拷贝的CGA-N12，其两侧是Lys-Arg对和两个Glu-Ala重复单元。高尔基体中的内源蛋白酶Kex2和Ste13识别并切除Lys-Arg和Glu-Ala对，分别从串联重复序列中释放短CGA-N12肽。CGA-N12肽已在巴斯德毕赤酵母中成功表达，通过HPLC测定，酵母培养物的收率高达30 mg / L。我们的研究表明，这项工作中采用的策略可能是直接在巴斯德毕赤酵母系统中表达小分子肽的好方法。高尔基体中的内源蛋白酶Kex2和Ste13识别并切除Lys-Arg和Glu-Ala对，分别从串联重复序列中释放短CGA-N12肽。CGA-N12肽已成功在巴斯德毕赤酵母中表达，通过HPLC测定，酵母培养物的收率高达30 mg / L。我们的研究表明，这项工作中采用的策略可能是直接在巴斯德毕赤酵母系统中表达小分子肽的好方法。高尔基体中的内源蛋白酶Kex2和Ste13识别并切除Lys-Arg和Glu-Ala对，分别从串联重复序列中释放短CGA-N12肽。CGA-N12肽已成功在巴斯德毕赤酵母中表达，通过HPLC测定，酵母培养物的收率高达30 mg / L。我们的研究表明，这项工作中采用的策略可能是直接在巴斯德毕赤酵母系统中表达小分子肽的好方法。