Yeast 3′,5′-bisphosphate nucleotidase: An affinity tag for protein purification

• Yeast 3′,5′-bisphosphate nucleotidase (HAL2) was purified and characterized.
• Kd for PAP was determined to be nanomolar with PAP analogue.
• Series of expression vectors with HAL2 as a tag were constructed.
• Purification protocol was designed and successfully used to purify proteins.
• HAL2 tag was compared with commonly used protein purification tags.

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 ∼0.3 μM and ∼11 s−1, respectively. Kd for PAP was 0.008 μM in the presence of Ca2+. pH was also found to affect interactions between HAL2 and PAP, with tightest binding (Kd ∼ 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′-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.