Background

The heterologous expression of human kinases in good purity and in a monomeric, soluble and active form can be challenging. Most of the reported successful attempts are carried out in insect cells as a host. The use of E. coli for expression is limited to a few kinases and usually is facilitated by large solubility tags that can limit biophysical studies and affect protein–protein interactions. In this report, we evaluate the methylotrophic yeast Pichia pastoris (P. pastoris) as a general-purpose host for expression of human kinases.

Methods

Six diverse kinases were chosen due to their therapeutic importance in human cancers. Tested proteins include serine/threonine kinases cyclin-dependent kinases 4 and 6 (CDK4 and 6) and aurora kinase A (AurKA), receptor tyrosine kinase erbB-2 (HER2), and dual specificity kinase mitogen-activated protein kinase kinase 3 (MKK3b). Noting that positively charged kinases expressed with higher yield, we sought to improve expression of two challenging targets, CDK6 and HER2, by fusing the highly basic, N-terminal domain of the secreted tyrosine-protein kinase VLK. The standard expression procedure for P. pastoris was adopted, followed by purification using affinity chromatography. Purity and activity of the proteins were confirmed and compared to published values.

Results

Some kinases were purified with good yield and purity and with comparable activity to commercially available versions. Addition of the VLK domain improved expression and decreased aggregation of CDK6 and HER2.

中文翻译：

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使用毕赤酵母作为通用宿主表达和纯化活性人激酶

背景

以高纯度和单体、可溶性和活性形式异源表达人类激酶可能具有挑战性。大多数报道的成功尝试都是在作为宿主的昆虫细胞中进行的。使用大肠杆菌进行表达仅限于少数激酶，并且通常由可限制生物物理研究并影响蛋白质-蛋白质相互作用的大溶解度标签促进。在本报告中，我们评估了甲基营养酵母毕赤酵母（P. pastoris）作为表达人类激酶的通用宿主。

方法

选择六种不同的激酶是因为它们在人类癌症中的治疗重要性。测试的蛋白质包括丝氨酸/苏氨酸激酶细胞周期蛋白依赖性激酶 4 和 6（CDK4 和 6）和极光激酶 A (AurKA)、受体酪氨酸激酶 erbB-2 (HER2) 和双特异性激酶丝裂原活化蛋白激酶激酶 3 (MKK3b) ）。注意到带正电荷的激酶以更高的产量表达，我们试图通过融合分泌型酪氨酸蛋白激酶 VLK 的高碱性 N 端结构域来改善两个具有挑战性的靶标 CDK6 和 HER2 的表达。采用毕赤酵母的标准表达程序，然后使用亲和层析进行纯化。确认蛋白质的纯度和活性，并与公布的值进行比较。

结果

一些激酶以良好的产量和纯度被纯化，并且具有与市售版本相当的活性。添加 VLK 结构域可改善 CDK6 和 HER2 的表达并减少其聚集。