title: Yeast 3',5'-bisphosphate nucleotidase: an affinity tag for protein purification.
abstract: 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.
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Ma
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Yilin
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Yanxing
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Yang
authors.affiliation: College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China. Electronic address: mhsun@zjnu.cn.
authors.forename: Meihao
authors.initials: M
authors.lastname: Sun
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006868:Hydrolysis; D009708:Nucleotidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D009708:Nucleotidases; C053354:bisphosphoadenylate 3'-nucleotidase
keywords: 3′,5′-Bisphosphate nucleotidase; Adenosine 5′-phosphosulfate kinase; Affinity chromatography; HAL2