裂殖壶菌可溶性蛋白的提取及双向电泳方法的建立

Extraction and Two-dimensional Electrophoresis Method Establishment of Soluble Protein from Schizochytriumsp FJU-512

裂殖壶菌Schizochytrium是目前用于商业化生产DHA的高产菌株之一。研究比对了多种裂殖壶菌可溶性蛋白的提取方法及纯化方式,并对IPG胶条的pH范围、胶条长度等双向电泳条件进行了优化。结果表明,裂殖壶菌可溶性蛋白的制备采用玻璃珠破碎结合TCA/丙酮沉淀的方法,并采用长度为24 cm,pH 3～10的IPG胶条进行等电聚焦效果最佳。并利用此电泳体系初步分析了37℃高温培养对裂殖壶菌可溶性蛋白表达的影响。

As one of the high-yield strains for DHA,Schizochytrium limacinum is currently used in commercial production. In order to establish a suitable two-dimensional electrophoresis used for screening the key proteins which regulate the production of DHA, many extraction and purification methods for soluble proteins from Schizochytrium limacinum were compared in the study. And the critical conditions of two-dimensional electrophoresis, including the pH range of IPG strips and the length of the strip, were opti- mized. The results showed that the soluble proteins of the strain could be prepared by the combined method of bead-milling and TCA/acetone precipitation,and that excellent two-dimentional electrophoresis results could be obtained by using the IPG strips with a length of 24 cm, pH 3 - 10 for isoelectric focusing. 2-DE maps showed the soluble proteins expression profile of Schizochytrium limacinum cultured at 37 ℃℃ changed obviously compared with those at 25 ℃, and 18 significantly altered spots were detected. Interestingly, one protein displaying twelve spots at 25 ℃, but they disappeared at the higher temperature （37 ℃ ）, which should be further researched. In conclusion,the results of the study have paved the way to further explore the regulation mechanism of DHA production by proteomic methodologies.