Hermanrud Christina E, Pathiraja Vimukthi, Matar Abraham, Duran-Struuck Raimon, Crepeau Rebecca L, Srinivasan Srimathi, Sachs David H, Huang Christene A, Wang Zhirui
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
Protein Expr Purif. 2012 Mar;82(1):70-4. doi: 10.1016/j.pep.2011.11.011. Epub 2011 Dec 1.
Yeast Pichia pastoris has been widely utilized to express heterologous recombinant proteins. P. pastoris expressed recombinant porcine interleukin 3 (IL3) has been used for porcine stem cell mobilization in allo-hematopoietic cell transplantation models and pig-to-primate xeno-hematopoietic cell transplantation models in our lab for many years. Since the yeast glycosylation mechanism is not exactly the same as those of other mammalian cells, P. pastoris expressed high-mannose glycoprotein porcine IL3 has been shown to result in a decreased serum half-life. Previously this was avoided by separation of the non-glycosylated porcine IL3 from the mixture of expressed glycosylated and non-glycosylated porcine IL3. However, this process was very inefficient and lead to a poor yield following purification. To overcome this problem, we engineered a non-N-glycosylated version of porcine IL3 by replacing the four potential N-glycosylation sites with four alanines. The codon-optimized non-N-glycosylated porcine IL3 gene was synthesized and expressed in P. pastoris. The expressed non-N-glycosylated porcine IL3 was captured using Ni-Sepharose 6 fast flow resin and further purified using strong anion exchange resin Poros 50 HQ. In vivo mobilization studies performed in our research facility demonstrated that the non-N-glycosylated porcine IL3 still keeps the original stem cell mobilization function.
酵母毕赤酵母已被广泛用于表达异源重组蛋白。多年来，我们实验室一直将毕赤酵母表达的重组猪白细胞介素3（IL3）用于异基因造血细胞移植模型和猪-灵长类动物异种造血细胞移植模型中的猪干细胞动员。由于酵母糖基化机制与其他哺乳动物细胞不完全相同，已证明毕赤酵母表达的高甘露糖糖蛋白猪IL3会导致血清半衰期缩短。以前，通过从表达的糖基化和非糖基化猪IL3混合物中分离出非糖基化猪IL3来避免这种情况。然而，这个过程效率非常低，纯化后产量也很低。为了克服这个问题，我们通过将四个潜在的N-糖基化位点替换为四个丙氨酸，构建了一个非N-糖基化版本的猪IL3。合成了密码子优化的非N-糖基化猪IL3基因，并在毕赤酵母中表达。使用Ni-Sepharose 6快速流动树脂捕获表达的非N-糖基化猪IL3，并使用强阴离子交换树脂Poros 50 HQ进一步纯化。在我们的研究机构进行的体内动员研究表明，非N-糖基化猪IL3仍然保持原始的干细胞动员功能。
