Abstract
Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake.
Highlights
In the post-genomic era, functional studies of genes rely in part on the expression and characterization of protein products of interest
We have previously described the expression of DNA ligase from bacteria phage T4 in soluble form as an Adenylate Kinase (AK) fusion protein using pAK-TAG cloning vector we developed [30] (Fig 1A). pAK-TAG expression vector contains multiple cloning sites at the C-terminus of AK followed by a stop codon for convenient in-frame fusion of any recombinant protein
The yield of recovery was estimated to be around 50% with over 7-fold purification for both AK-TNFα (Fig 2) and AK-T-TNFα (Fig 3) fusion proteins based on the increase in specific activity of AK (Table 1)
Summary
In the post-genomic era, functional studies of genes rely in part on the expression and characterization of protein products of interest. To this end, the concurrent use of fusion tags with DNA cloning technology has become a routine practice in recombinant protein expression and purification [1–4]. Whether large or small, can often impede upon the structure and functions of a target protein expressed and may need to be removed during or after purification [6, 10, 14, 15]. A specific proteolytic cleavage site is often introduced between the tag and the protein of PLOS ONE | DOI:10.1371/journal.pone.0156106. A specific proteolytic cleavage site is often introduced between the tag and the protein of PLOS ONE | DOI:10.1371/journal.pone.0156106 May 23, 2016
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