High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG
May
High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG
Dan Luo 0 1
Caixia Wen 0 1
Rongchuan Zhao 0 1
Xinyu Liu 0 1
Xinxin Liu 0 1
Jingjing Cui 0 1
Joshua G. Liang 1
Peng Liang 0 1
0 Department of Biochemistry & Molecular Biology, School of Life Sciences, Sichuan University , Chengdu, China, 2 Clover Biopharmaceuticals, Chengdu, China, 3 GenHunter Corporation, Grassmere Park, Nashville , United States of America
1 Editor: Bostjan Kobe, University of Queensland , AUSTRALIA
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.
Data Availability Statement; All relevant data are within the paper
-
OPEN ACCESS
Funding: This work was supported in part by a
National 863 grant (2012AA02A305) (PL) and a grant
(2012ZX09103301) from the Chinese Ministry of
Science and Technology (PL), and a 973 grant
(2012CB910700) from the Chinese Ministry of
Education (PL). These authors (PL and JGL) are
affiliated with commercial companies: Clover
Biopharmaceuticals and GenHunter Corporation. The
funders provided support in the form of salaries for
authors PL and JGL, but did not have any additional
role in the study design, data collection and analysis,
Introduction
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
]. Although numerous affinity tags have been developed over the years to
facilitate the expression and purification of recombinant proteins from Escherichia coli [
5–10
],
Glutathione S-transferase (GST-Tag) [
11
], Maltose-binding protein (MBP-Tag) [
12
] and
6xHIS-Tag [
13
] remain the most popular methods of choice due to the commercially available
expression vectors and their downstream purification systems.
All tags, 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
].
Thus, a specific proteolytic cleavage site is often introduced between the tag and the protein of
decision to publish, or preparation of the manuscript.
The specific roles of these authors are articulated in
the ‘author contributions’ section.
Competing Interests: These authors (PL and JGL)
are affiliated with commercial companies: Clover
Biopharmaceuticals and GenHunter Corporation. On
behalf of all authors, the corresponding author
declares that commercial affiliations do not alter the
adherence to PLOS ONE policies on sharing data.
interested to be expressed on an expression vector. Among many site specific proteases used
for the cleavage of the tags’ off target proteins [
7, 16, 17
], thrombin is the most widely used due
to its high target sequence-specificity and rarity found in natural proteins [
18–21
].
Despite the wide-spread use of these tailor-made expression vectors and purification
strategies, frustrations often occur when a target protein is expressed either at low level, or as
insoluble inclusion bodies [
5, 22
]. Although His-Tag may allow purification of insoluble proteins
after complete unfolding with a denaturant, the yield of refolding and full recovery of biological
functions of a recombinant protein may be less predictable [23]. In addition, depending on the
level of expression, the yield and purity of a recombinant protein from a single affinity column
can be far from perfect due to endogenous host cell proteins bound to the columns.
Adenylate Kinase (AK) catalyzes the enzymatic conversion of AMP to ADP using ATP:
Mg2+ATP + AMP $ Mg2+ADP + ADP, and is an essential enzyme in all living organisms [
24,
25
]. Previous work from our laboratory as well as by others showed that (...truncated)