Yang Yang, Ma Jianhui, Yang Yilin, Zhang Xiao, Wang Yanxing, Yang Ling, Sun Meihao
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
Protein Expr Purif. 2014 May;97:81-7. doi: 10.1016/j.pep.2014.02.014. Epub 2014 Mar 6.
Affinity chromatography is one of the most popular methods for protein purification. Each tag method has its advantages and disadvantages, and combination of different tags and developing of new tags had been proposed and performed. Yeast 3',5'-bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3'-phosphoadenosine 5'-phosphate (PAP) with submicromolar Km, which indicated the tight interactions between HAL2 and PAP. In order to explore the feasibility of HAL2 as a protein purification affinity tag, HAL2 was further characterized with PAP as substrate. Results demonstrated that KmPAP and kcatPAP were ∼0.3μM and ∼11s(-)(1), respectively. Kd for PAP was 0.008μM in the presence of Ca(2+). pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd∼8nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca(2+), which could satisfy the metal requirement for PAP binding, prevent hydrolysis of immobilized PAP and could be chelated by ethylene glycol tetraacetic acid (EGTA) for elution. A series of expression vectors were further constructed and Escherichia coli adenosine 5'-phosphosulfate kinase (APSK) was prokaryotically expressed, purified and characterized. Ready to use expression vector with eight commonly used restriction enzyme recognition sites in multiple cloning site was subsequently constructed. By comparing with current popular tags, HAL2 was found to be an efficient and economical tag for prokaryotic protein expression and purification.
亲和层析是蛋白质纯化最常用的方法之一。每种标签方法都有其优缺点，不同标签的组合以及新标签的开发已被提出并实施。酵母3',5'-二磷酸核苷酸酶，也称为HAL2，以亚微摩尔级的Km水解3'-磷酸腺苷5'-磷酸（PAP），这表明HAL2与PAP之间存在紧密的相互作用。为了探索HAL2作为蛋白质纯化亲和标签的可行性，以PAP为底物对HAL2进行了进一步表征。结果表明，KmPAP和kcatPAP分别约为0.3μM和约11s(-1)。在Ca(2+)存在下，PAP的Kd为0.008μM。还发现pH会影响HAL2与PAP之间的相互作用，在pH 7.5和8时结合最紧密（Kd约为8nM）。基于在Ca(2+)存在下对PAP琼脂糖的纳摩尔亲和力合理设计了纯化方案，该方案可以满足PAP结合的金属需求，防止固定化PAP的水解，并且可以被乙二醇四乙酸（EGTA）螯合用于洗脱。进一步构建了一系列表达载体，并对大肠杆菌腺苷5'-磷酸硫酸激酶（APSK）进行了原核表达、纯化和表征。随后构建了在多克隆位点具有八个常用限制酶识别位点的即用型表达载体。通过与当前流行的标签进行比较，发现HAL2是用于原核蛋白质表达和纯化的一种高效且经济的标签。
