Calcagno Sarah, Klein Christian D
Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, Heidelberg University, Im Neuenheimer Feld 364, 69120, Heidelberg, Germany.
Appl Microbiol Biotechnol. 2016 Aug;100(16):7091-102. doi: 10.1007/s00253-016-7470-3. Epub 2016 Mar 29.
The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins.
恶性疟原虫的甲硫氨酸氨肽酶1b（PfMetAP 1b）被克隆、在大肠杆菌中表达并进行了表征。令人惊讶的是，与其他需要钴等重金属辅因子的甲硫氨酸氨肽酶（MetAPs）不同，该酶能被锌离子可靠地激活。通过His标签金属螯合到亚氨基二乙酸琼脂糖以及通过与氯乙酰胺己基琼脂糖共价结合，可以实现该酶的固定化。共价固定化的酶表现出长期稳定性，能够对N端甲硫氨酸进行连续的异相处理，例如在重组蛋白中。与其他MetAPs使用钴不同，锌激活避免了将具有毒理学风险和氧化潜力的重金属引入生物技术过程。因此，PfMetAP 1b是重组蛋白酶促翻译后加工的有用工具。
