Yeast 3鈥?5鈥?bisphosphate nucleotidase: An affinity tag for protein purification

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• 作者：Yang Yang ; Jianhui Ma ; Yilin Yang ; Xiao Zhang ; Yanxing Wang¹ ; Ling Yang ; Meihao Sun ; ^{mhsun@zjnu.cn" class="auth_mail}
• 关键词：Affinity chromatography ; 3&prime ; 5&prime ; -Bisphosphate nucleotidase ; Adenosine 5&prime ; -phosphosulfate kinase ; HAL2
• 刊名：Protein Expression and Purification
• 出版年：May, 2014
• 年：2014
• 卷：97
• 期：Complete
• 页码：81-87
• 全文大小：833 K

文摘

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鈥?5鈥?bisphosphate nucleotidase, also known as HAL2, hydrolyzes 3鈥?phosphoadenosine 5鈥?phosphate (PAP) with submicromolar K_m, 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 K_mPAP and k_catPAP were 鈭?.3 渭M and 鈭?1 s^{鈭?/sup>1, respectively. K_d for PAP was 0.008 渭M in the presence of Ca2+. pH was also found to affect interactions between HAL2 and PAP, with tightest binding (K_d 鈭?#xA0;8 nM) at pH 7.5 and 8. The purification protocol was rationally designed based on nanomolar affinity to PAP agarose in the presence of Ca2+, 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鈥?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.}