Small Molecule Discovery Center, Department of Pharmaceutical Chemistry and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
Small Molecule Discovery Center, Department of Pharmaceutical Chemistry and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA.
Protein Expr Purif. 2021 Mar;179:105780. doi: 10.1016/j.pep.2020.105780. Epub 2020 Oct 25.
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.
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.
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.
以单体、可溶性和活性形式高效表达人源激酶具有一定的挑战性。大多数已报道的成功尝试都是在昆虫细胞中作为宿主进行的。在大肠杆菌中进行表达的方法通常局限于少数几种激酶，而且通常需要使用大的可溶性标签，这可能会限制生物物理研究并影响蛋白质-蛋白质相互作用。在本报告中，我们评估了甲醇营养型酵母巴斯德毕赤酵母（Pichia pastoris）作为表达人源激酶的通用宿主。
选择了六种不同的激酶，因为它们在人类癌症中的治疗意义重大。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）和双重特异性激酶丝裂原激活蛋白激酶激酶 3（MKK3b）。鉴于带正电荷的激酶表达产量更高，我们试图通过融合分泌型酪氨酸蛋白激酶 VLK 的高度碱性 N 端结构域来提高两个具有挑战性的靶标 CDK6 和 HER2 的表达。采用巴斯德毕赤酵母的标准表达程序，然后通过亲和层析进行纯化。对蛋白质的纯度和活性进行了确认，并与已发表的值进行了比较。
一些激酶的表达产量和纯度较高，且具有与市售版本相当的活性。添加 VLK 结构域可提高 CDK6 和 HER2 的表达水平并减少聚集。
