Accepted Manuscript

High-level expression of soluble recombinant proteins in Escherichia coli using an

HE-maltotriose-binding protein fusion tag

Yingqian Han, Wanying Guo, Bingqian Su, Yujie Guo, Jiang Wang, Beibei Chu,
Guoyu Yang

PII: S1046-5928(17)30395-9
DOI: 10.1016/j.pep.2017.09.013
Reference: YPREP 5165

To appear in: Protein Expression and Purification

Received Date: 4 July 2017

Revised Date: 25 September 2017
Accepted Date: 25 September 2017

Please cite this article as: Y. Han, W. Guo, B. Su, Y. Guo, J. Wang, B. Chu, G. Yang, High-level
expression of soluble recombinant proteins in Escherichia coli using an HE-maltotriose-binding protein
fusion tag, Protein Expression and Purification (2017), doi: 10.1016/j.pep.2017.09.013.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT
High-level expression of soluble recombinant proteins in Escherichia coli using an HE-
maltotriose-binding protein fusion tag

Yingqian Hana#, Wanying Guoa#, Bingqian Sua, Yujie Guoa, Jiang Wanga, Beibei Chua, Guoyu
Yanga∗

a College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou

PT
450002, Henan Province, PR China
# These authors contributed equally to this work.

RI
∗Corresponding authors at: Henan Agricultural University, College of Animal Science and
Veterinary Medicine, No. 95, Wenhua Road, Zhengzhou 450002, HenanProvince, PR China.
Tel.: +86 371 6355 8189;

SC
fax: +86 371 6355 8180.
E-mail addresses: haubiochem@163.com.

Abstract
U
AN
Recombinant proteins are commonly expressed in prokaryotic expression systems for large-scale
production. The use of genetically engineered affinity and solubility enhancing fusion proteins has
M

increased greatly in recent years, and there now exists a considerable repertoire of these that can
be used to enhance the expression, stability, solubility, folding, and purification of their fusion
partner. Here, a modified histidine tag (HE) used as an affinity tag was employed together with a
D

truncated maltotriose-binding protein (MBP; consisting of residues 59–433) from Pyrococcus

TE

furiosus as a solubility enhancing tag accompanying a tobacco etch virus protease-recognition site
for protein expression and purification in Escherichia coli. Various proteins tagged at the
N-terminus with HE-MBP(Pyr) were expressed in E. coli BL21(DE3) cells to determine
EP

expression and solubility relative to those tagged with His6-MBP or His6-MBP(Pyr). Furthermore,
four HE-MBP(Pyr)-fused proteins were purified by immobilized metal affinity chromatography to
assess the affinity of HE with immobilized Ni2+. Our results showed that HE-MBP(Pyr) represents
C

an attractive fusion protein allowing high levels of soluble expression and purification of
AC

recombinant protein in E. coli.

Keywords: maltotriose binding protein; histiding tag; recombinant protein; soluble protein
expression

1. Introduction

As a product of DNA recombinant technology, fusion proteins expressed in several host

organisms have been developed as a class of novel biomolecules with multifunctional properties
and comprehensive applications in biological research [1]. Compared with eukaryotic expression
 ACCEPTED MANUSCRIPT
systems, Escherichia coli remains the dominant host for producing recombinant proteins due to its
advantageous, rapid, inexpensive, and high-yield protein production, together with its
well-characterized genetics and variety of available molecular tools [2, 3]. However, protein
insolubility, conformation, stability, and structural flexibility, as well as low purification yields and
host-cell toxicity, remain challenges that must be resolved when the E. coli bacterial system is
used to express recombinant proteins [4]. Despite the aforementioned issues associated with E.
coli recombinant-protein production, the cost benefits, ease of use, and scale make it essential to

PT
design new strategies for the production of recombinant soluble protein in this host [3].

Fusion tags are proteins or peptide molecules capable of soluble expression in E. coli and

RI
commonly used to facilitate target-protein expression, resistance to proteolytic degradation,
solubility, and purification [5-7]. Widely used fusion tags can be divided into affinity tags and

SC
solubility tags. Affinity tags used for protein isolation and purification include hexahistidine
(His6), maltose-binding protein (MBP)[8], glutathione-S-transferase) [9], and Strep-tag II [10].
Solubility tags for soluble-protein production include MBP[11], N-utilization substance A [12],

U
thioredoxin A [13], and small ubiquitin-related modifier (SUMO)[5]. Some protein tags, such as
glutathione-S-transferase and MBP, can also function as promoters of both affinity and solubility.
AN
Additionally, numerous solubility enhancing tags are employed with affinity tags to enhance
protein solubility and yield and simplify the purification procedure. Protease-cleavage sites
M

located between the fusion partner and target protein are invariably employed to remove the fusion
partner from the final protein to counteract potential interference with attainment of proper
structure and function by the target protein [3]. Nevertheless, the effectiveness of a fusion protein
D

is not always a given for every protein target [14], and many proteins frustratingly remain
TE

insoluble or difficult to purify regardless of which of these fusions are used. For example, a
His-tag used as a fusion partner for protein expression affects the formation of inclusion bodies
[15]. Therefore, new effective tags are needed to increase the accumulation of soluble recombinant
EP

proteins expressed in E. coli while also assisting purification.

The HEHEHE-tag is a modified hexahistidine tag in which every second histidine residue is
C

replaced by a more hydrophilic glutamate, which still allows efficient purification by immobilized
metal ion affinity chromatography (IMAC) [16]. It is plausible that substitution of a His-tag with a
AC

HEHEHE-tag could represent an effective strategy for addressing the issue of inclusion bodies
formed by recombinant proteins. In this study, hence, an (HE)7-tag used as an affinity tag was
incorporated with maltotriose-binding protein, a presumptive solubility tag isolated from
Pyrococcus furiosus (GenBank accession no. WP_011013078) [MBP(Pyr)], and located adjacent
to a tobacco etch virus (TEV) protease-recognition site as a new fusion tag [HE-MBP(Pyr)] for
protein expression and purification in E. coli. His6-MBP [17] was used to compare the expression
and purification yields of HE-MBP(Pyr) when fused to the protein of interest.

2. Materials and methods

 ACCEPTED MANUSCRIPT
2.1 . Strains, vectors, and reagents

E. coli strain BL21(DE3), previously preserved in our laboratory, was used for cloning and
expression. The plasmids used for expression (Table 1) were preserved or constructed in our
laboratory. The pUC57-HE-MBP(Pyr)-Nb plasmid was synthesized by GenScript (Nanjing,
China). Restriction endonucleases and PageRuler pre-stained protein ladder were purchased from
Thermo Fisher Scientific (Waltham, MA, USA). The SanPrep column plasmid mini-prep kit,
SanPrep column DNA gel extraction kit, and SanPrep column PCR product purification kit were

PT
purchased from Sangon (Shanghai, China). T4 DNA ligase was purchased from Takara Bio
(Dalian, China). High-affinity Ni-NTA resin was obtained from GenScript.

RI
2.2. Construction and verification of recombinant plasmids

SC
The pUC57-HE-MBP(Pyr)-Nb and pET-21b vectors were digested with NdeI/XhoI and
ligated to obtain the pET21b-HE-MBP(Pyr)-Nb recombinant plasmid as the carrier for other
recombinant plasmids. Further, HE-MBP(Pyr) was replaced with either His6-MBP(Pyr) or

U
His6-MBP. The gene fragments encoding H5HA10, PCV2b, NLSFMD, HAFnt, Cago60,
AN
PCV2VHHC3, CdiGMP499 (Caulobacter vibrioides), CdiGMP026 (Phenylobacterium zucineum),
Prop acid OAS, and FMDV98, respectively, were digested with BamHI/XhoI and inserted into the
BamHI/XhoI-digested pET21b-HE-MBP(Pyr), pET21b-His6-MBP(Pyr), and pET21b-His6-MBP
M

vectors. All recombinant plasmids were transformed into E. coli strain BL21(DE3) cells. Positive
clones were identified by restriction endonuclease reaction and verified by DNA sequencing.
D
TE

2.3. Protein expression and solubility identification

Verified transformants were cultured overnight at 37°C and diluted 1:100 in Luria–Bertani
broth culture containing 100 µg/mL ampicillin. When the optical density at 600 nm reached 0.6,
EP

0.5 mM of isopropyl β-D-thiogalactopyranoside (IPTG) was added to induce protein expression

for 12 h to 16 h at 25°C. Cells were collected by 8 000 g 10 min and resuspended in
C

phosphate-buffered saline (pH 7.4), followed by disruption by sonication on ice after

ultracentrifugation for 20 min at 4°C. The soluble and insoluble fractions of recombinant proteins
AC

were analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
under denaturing conditions to appraise the relative expression level and solubility of each fusion
protein. Expression and solubility levels were determined using an Amersham Imager 600 (GE
Healthcare, Pittsburgh, PA, USA) and calculated as described previously [18].

2.4. Protein purification

The induced cells were harvested by 8 000 g 10 min at 4°C and dissolved in ice-cold lysis
buffer [50 mM Na2HPO4, 300 mM NaCl, and 10 mM imidazole (pH 8.0)]. The supernatant
 ACCEPTED MANUSCRIPT
obtained by sonication following ultracentrifugation was incubated for 1 h at 4°C with shaking at
200 rpm along with pre-equilibrated Ni-NTA resin. The resin was allowed to settle completely by
gravity, and aliquots of the supernatant were aspirated. Buffers containing gradient concentrations
of imidazole were used at 10-fold the column volume to elute proteins bound to the Ni-NTA resin.
The eluate was collected and analyzed by 12% SDS-PAGE under denaturing conditions to
evaluate the affinity of the HE tag relative to the His6 tag.

PT
3. Results
3.1. Construction of the recombinant plasmids

RI
To test the effects of the HE-MBP(Pyr) tag on the expression and solubility of Nb, H5HA10,
PCV2b, NLSFMD, HAFnt, Cago60, PCV2VHHC3, CdiGMP499 (C. vibrioides), CdiGMP026 (P.

SC
zucineum), Prop acid OAS, and FMDV98, the gene sequences were digested and ligated into
similarly digested pET21b-HE-MBP(Pyr), pET21b-His6-MBP, and pET21b-His6-MBP(Pyr)
vectors (Fig. 1). The HE tag represents a modified His-based tag containing 7×

U
histidine-glutamate repeats (HEHEHEHEHEHEHE) capable of IMAC purification [16]. The
MBP(Pyr) tag consists of a maltotriose-binding protein derived from P. furiosus (GenBank
AN
accession no. WP_011013078), and the nucleic acid sequence of the combined HE-MBP(Pyr) tag
was codon-optimized for expression in E coli. The TEV protease-recognition site was inserted
M

adjacent to the HE-MBP(Pyr) tag to enable efficient separation during purification.

3.2. Protein expression and solubility identification

D

The constructed plasmids were transformed into E. coli BL21(DE3) cells, and expression was
TE

driven by the IPTG-inducible T7 promotor. All fusion proteins expressed in BL21(DE3) cells were
identified by SDS-PAGE analysis of their soluble and insoluble fractions. Our results showed that
the expression level and solubility of target proteins tagged with HE-MBP(Pyr) were exceeded
EP

those fused with other tags. Furthermore, HE-MBP(Pyr) fused with target proteins exhibiting low
levels of expression allowed increased expression levels accompanied by high abundance in the
C

soluble fraction, which was consistent with target proteins exclusively expressed in the form of
inclusion bodies (Fig. 2). Additionally, we observed that the expression level and solubility of nine
AC

different target proteins were markedly improved by the HE-MBP(Pyr) fusion as compared with
levels observed by fusion with His6-MBP (Fig. 3 and Table 1).

To delineate the variation in protein expression observed between MBP(Pyr) and MBP
fusions, five target proteins were tagged with His6-MBP(Pyr) or His6-MBP. Our results showed
that the protein content in the soluble and insoluble fractions was represented by new bands
appearing at the correct molecular weights relative to controls derived from uninduced E. coli
cells. The expression levels of Cago60 and FMDV98 tagged with MBP(Pyr) were comparable
with those tagged with MBP. Although levels of MBP(Pyr)-H5HA10, -HAFnt, -Prop acid OAS
 ACCEPTED MANUSCRIPT
were lower relative to MBP-tagged variants, increased soluble-protein production was detected in
the MBP(Pyr)-tagged variants as compared with the MBP-tagged proteins (Table 2). Notably,
MBP(Pyr)-HAFnt was only observed in the soluble fraction (Fig. S1).

To further evaluate variations in expression between HE- and His6-tagged fusions, seven
proteins were fused with HE-MBP(Pyr) or His6-MBP(Pyr). Despite the accumulation of
significant levels of HE-MBP(Pyr)-PCV2VHHC3, -CdiGMP499, and -NLSFMD, SDS-PAGE
analysis showed that the accumulation of proteins fused with HE was similar to those fused with

PT
His6 (Fig. S2). However, the solubility of all HE-tagged proteins was higher relative to
His6-tagged proteins (Table 3).

RI
3.3. Protein purification

SC
To assess the affinity of HE-tagged proteins to immobilized Ni2+, HE-MBP(Pyr)-Nb,
-H5HA10, -Cago60, -FMDV98, and -Prop acid OAS, as well as His6-MBP(Pyr)-H5HA10,

U
-Cago60, -FMDV98, and -Prop acid OAS recombinant proteins were mixed gently with Ni-NTA
resin equilibrated with lysis buffer at 4°C for 60 min, followed by elution using an imidazole
AN
gradient. Gravity collection of the supernatant and SDS-PAGE analysis showed higher yields of
purified HE-tagged proteins as compared with His6-tagged proteins (Fig. S3).
M

4. Discussion
D

E. coli is an important host for recombinant-protein expression due to its easy manipulation
and high biomass-to-cost ratio. However, some heterologous proteins produced in E. coli form
TE

aggregates of insoluble folding intermediates known as inclusion bodies, which represent a major
obstacle for protein purification and further functional research. Protein-fusion technology has
facilitated increased yields of soluble, well-folded, functional proteins, with numerous solubility
EP

or affinity tags of either natural origin or artificial design having been exploited to enhance protein
solubility and yield and facilitate purification [7]. However, the effectiveness of a fusion construct
C

to enhance target-protein expression or solubility may not always be a given. Therefore, new tags
are needed to increase the accumulation of soluble recombinant proteins expressed in E. coli while
AC

also assisting their successful purification. Currently, proteins, such as Fh8 [3], XTEN [19], SmbP
[14], and GAPDH [20], are exploited to develop expression and/or solubility systems.

In this study, a new fusion tag [HE-MBP(Pyr)]was developed and applied to improve the
solubility of recombinant proteins expressed in E. coli. Our results revealed that HE-MBP(Pyr)
facilitated high-levels of soluble expression of target proteins previously exhibiting low levels of
expression or expression as inclusion bodies (Fig. 2). To validate the efficacy of HE-MBP(Pyr),
the expression of 11 target proteins, including monoclonal antibodies [Nb [21] and PCV2VHHC3
(GenBank accession no. AGV76522.1)], antigen proteins [H5HA10, PCV2b capsid (GenBank
 ACCEPTED MANUSCRIPT
accession no. ABV21950.1), NLSFMD, HAFnt, and FMDV98 (GenBank accession no.
AGO58328.1)], a polymeric protein [Cago60 [22]], and a cyclic dinucleotide synthetase
[CdiGMP499 (GenBank accession no. AED89582.1), Prop acid OAS (GenBank accession no.
WP_041707532.1), and CdiGMP026 (GenBank accession no. WP_012523835)], tagged with
HE-MBP(Pyr) were compared with that of the same proteins tagged with His6-MBP, a common
fusion tag used for increasing production and solubility of its fusion partner. Our results shown in
Figure 3 indicated a ~19-fold difference in the number of HE-MBP(Pyr)-tagged proteins observed

PT
in the soluble fraction relative to His6-MBP-tagged proteins. Consistent with this result, we
observed a 4-fold increase in the number of soluble HE-MBP(Pyr)-tagged fusion proteins as

RI
compared with soluble His6-MBP-fused proteins, suggesting that HE-MBP(Pyr) improved the
solubility of its fusion proteins.

SC
MBP(Pyr) is a truncated maltotriose-binding protein (consisting of residues 59–433) from P.
furiosus and belonging to the bacterial solute-binding protein-1 family. Interestingly, the solubility
of MBP(Pyr) predicted by PROSO II [23] is higher than that of MBP, the product of the malE

U
gene from E. coli and a member of the same protein family. MBP acts as a receptor for chemotaxis
and gene regulation and represents one of the oldest and most popular fusion partners utilized for
AN
recombinant-protein producing bacterial systems [24, 25]. Here, we examined whether MBP(Pyr)
was superior to MBP for the expression and solubility of heterologous proteins expressed in E.
M

coli. We observed that the solubility of His6-MBP(Pyr)-fusion proteins was uniformly higher than
that observed for His6-MBP-fusion proteins (Table 2). However, the relative expression level of
His6-MBP-fusion proteins was lower than that observed for His6-MBP(Pyr)-fusion proteins,
D

possibly a result of the His6 tag. This was supported by the high levels of expression observed
TE

for HE-MBP(Pyr)-H5HA10, -Cago60, -FMDV98, and -Prop acid OAS in E. coli as compared
with the His6-MBP(Pyr)-tagged variants (Tables 1 and 2). These results supported that
HE-MBP(Pyr) might represent an effective fusion tag enabling high-levels of soluble heterologous
EP

protein expression in E. coli.

To demonstrate the efficacy of the HE-MBP(Pyr) tag, HE-MBP(Pyr)-fusion-protein

C

expression, solubility, and purification were compared with those of His6-MBP(Pyr)-tagged

proteins. Our results revealed higher expression and solubility of HE-MBP(Pyr)-fusion proteins
AC

relative to His6-MBP(Pyr)-fusion proteins. Additionally, investigation of the ability of HE-tagged

proteins to be purified by IMAC using Ni-NTA resin revealed prominent SDS-PAGE-derived
bands associated with the HE-MBP(Pyr)-fused proteins from the elution fraction as compared
with those observed from His6-MBP(Pyr)-fused proteins (Fig. S3), suggesting that the
HE-MBP(Pyr)-fusion tag improved soluble expression and purification of heterologous protein in
E. coli. Furthermore, we also showed that the HE-tag reduced the formation of inclusion bodies by
recombinant proteins. Further studies investigating HE-tagged proteins for purification are needed
to elucidate the specific characteristics associated with HE-MBP(Pyr) fusion on enhancing the
solubility of target proteins.
 ACCEPTED MANUSCRIPT
In summary, use of the HE-MBP(Pyr)-fusion tag increased the relative expression and
solubility of heterologous proteins in E. coli as compared with use of the His6-MBP and
His6-MBP(Pyr) tags. Additionally, we verified that HE-MBP(Pyr)-fused proteins were capable of
being purified by IMAC, and that incorporation of a TEV protease-recognition site allowed tag
separation during purification. Our results demonstrated that HE-MBP(Pyr) represents an
attractive fusion construct for use in the purification of high levels of soluble recombinant proteins
expressed in E. coli.

PT
Acknowledgements
This work was supported by grants from the Ministry of Agriculture of China (grant no. 2011-G35;

RI
2014ZX0801015B).

SC
References
[1] X.Y. Chen, J.L. Zaro, W.C. Shen, Fusion protein linkers: Property, design and functionality,

U
Advanced Drug Delivery Reviews 65 (2013) 1357-1369.
AN
[2] A.L. Demain, P. Vaishnav, Production of recombinant proteins by microbes and higher
organisms, Biotechnol Adv 27 (2009) 297-306.
[3] S. Costa, A. Almeida, A. Castro, L. Domingues, Fusion tags for protein solubility, purification
and immunogenicity in Escherichia coli: the novel Fh8 system, Front Microbiol 5 (2014) 63.
M

[4] C.L. Young, Z.T. Britton, A.S. Robinson, Recombinant protein expression and purification: a
comprehensive review of affinity tags and microbial applications, Biotechnol J 7 (2012) 620-634.
D

[5] T.R. Butt, S.C. Edavettal, J.P. Hall, M.R. Mattern, SUMO fusion technology for
difficult-to-express proteins, Protein Expr Purif 43 (2005) 1-9.
TE

[6] T. Panavas, C. Sanders, T.R. Butt, SUMO fusion technology for enhanced protein production
in prokaryotic and eukaryotic expression systems, Methods Mol Biol 497 (2009) 303-317.
[7] D. Walls, S.T. Loughran, Tagging recombinant proteins to enhance solubility and aid
EP

purification, Methods Mol Biol 681 (2011) 151-175.

[8] K.D. Pryor, B. Leiting, High-level expression of soluble protein in Escherichia coli using a
His6-tag and maltose-binding-protein double-affinity fusion system, Protein Expr Purif 10 (1997)
C

309-319.
[9] D.B. Smith, K.S. Johnson, Single-step purification of polypeptides expressed in Escherichia
AC

coli as fusions with glutathione S-transferase, Gene 67 (1988) 31-40.

[10] T.G. Schmidt, A. Skerra, One-step affinity purification of bacterially produced proteins by
means of the "Strep tag" and immobilized recombinant core streptavidin, J Chromatogr A 676
(1994) 337-345.
[11] R.B. Kapust, D.S. Waugh, Escherichia coli maltose-binding protein is uncommonly effective
at promoting the solubility of polypeptides to which it is fused, Protein Sci 8 (1999) 1668-1674.
[12] V. De Marco, G. Stier, S. Blandin, A. de Marco, The solubility and stability of recombinant
proteins are increased by their fusion to NusA, Biochem Biophys Res Commun 322 (2004)
766-771.
[13] T. Yasukawa, C. Kanei-Ishii, T. Maekawa, J. Fujimoto, T. Yamamoto, S. Ishii, Increase of
 ACCEPTED MANUSCRIPT
solubility of foreign proteins in Escherichia coli by coproduction of the bacterial thioredoxin, J
Biol Chem 270 (1995) 25328-25331.
[14] T. Vargas-Cortez, J.R. Morones-Ramirez, I. Balderas-Renteria, X. Zarate, Expression and
purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein
from Nitrosomonas europaea, Protein Expr Purif 118 (2016) 49-54.
[15] S. Zhu, C. Gong, L. Ren, X. Li, D. Song, G. Zheng, A simple and effective strategy for
solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions
enhance soluble expression, Appl Microbiol Biotechnol 97 (2013) 837-845.

PT
[16] V. Tolmachev, C. Hofstrom, J. Malmberg, S. Ahlgren, S.J. Hosseinimehr, M. Sandstrom, L.
Abrahmsen, A. Orlova, T. Graslund, HEHEHE-tagged affibody molecule may be purified by
IMAC, is conveniently labeled with [(9)(9)(m)Tc(CO)(3)](+), and shows improved biodistribution

RI
with reduced hepatic radioactivity accumulation, Bioconjug Chem 21 (2010) 2013-2022.
[17] C.J. Rocco, K.L. Dennison, V.A. Klenchin, I. Rayment, J.C. Escalante-Semerena,
Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from

SC
Escherichia coli, Plasmid 59 (2008) 231-237.
[18] M.T. Nguyen, M. Krupa, B.K. Koo, J.A. Song, T.T. Vu, B.H. Do, A.N. Nguyen, T. Seo, J. Yoo,
B. Jeong, J. Jin, K.J. Lee, H.B. Oh, H. Choe, Prokaryotic Soluble Overexpression and Purification

U
of Human VEGF165 by Fusion to a Maltose Binding Protein Tag, PLoS One 11 (2016) e0156296.
AN
[19] S. Ding, M. Song, B.C. Sim, C. Gu, V.N. Podust, C.W. Wang, B. McLaughlin, T.P. Shah, R.
Lax, R. Gast, R. Sharan, A. Vasek, M.A. Hartman, C. Deniston, P. Srinivas, V. Schellenberger,
Multivalent antiviral XTEN-peptide conjugates with long in vivo half-life and enhanced solubility,
Bioconjug Chem 25 (2014) 1351-1359.
M

[20] Z. Chen, Y. Li, X. Sun, Q. Yuan, Improvement of expression level of polysaccharide lyases
with new tag GAPDH in E. coli, J Biotechnol 236 (2016) 159-165.
D

[21] H. Liu, Y. Wang, H. Duan, A. Zhang, C. Liang, J. Gao, C. Zhang, B. Huang, Q. Li, N. Li, S.
Xiao, E.M. Zhou, An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells
TE

inhibits porcine reproductive and respiratory syndrome virus replication, Vet Microbiol 181 (2015)
252-260.
[22] Y. Hsia, J.B. Bale, S. Gonen, D. Shi, W. Sheffler, K.K. Fong, U. Nattermann, C. Xu, P.S.
EP

Huang, R. Ravichandran, S. Yi, T.N. Davis, T. Gonen, N.P. King, D. Baker, Design of a
hyperstable 60-subunit protein icosahedron, Nature 535 (2016) 136-139.
[23] P. Smialowski, G. Doose, P. Torkler, S. Kaufmann, D. Frishman, PROSO II--a new method
C

for protein solubility prediction, Febs j 279 (2012) 2192-2200.

[24] M. Lebendiker, T. Danieli, Purification of Proteins Fused to Maltose-Binding Protein,
AC

Methods Mol Biol 1485 (2017) 257-273.

[25] H. Nikaido, Maltose transport system of Escherichia coli: an ABC-type transporter, FEBS
Lett 346 (1994) 55-58.
 ACCEPTED MANUSCRIPT
Figure captions

PT
RI
U SC
AN
Figure 1. Schematic representation of expression vectors. All the recombinant gene segments were
inserted into the pET21b vector following restriction-enzyme digestion and ligation.
MBP(Pyr)-TEV: gene sequence encodes maltotriose-binding protein and TEV
M

protease-recognition site. MBP-TEV: gene sequence encodes maltose-binding protein and TEV
protease-recognition site. Expression of the fusion proteins in E. coli was driven by the
IPTG-inducible T7 promoter, with ampicillin as the selection marker.
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE

Figure 2 SDS-PAGE analysis of expression of HE-MBP(Pyr)-fused recombinant proteins.

Expression was induced by 0.5 mM IPTG at 25°C. Expression levels were compared against those
EP

of proteins expressed with other fusion tags. LSL150: a lectin-based affinity tag. Cherry: a red
fluorescent protein used as a solubility tag. His6: hexa-histidine affinity tag. HAFnt, NLSFMD,
PCV2b, and H5HA10 were target proteins. Arrows indicate the target fusion proteins. M: protein
C

molecular-weight marker; C: total cellular protein level before IPTG induction (negative control);
AC

I: total cellular protein level after IPTG induction; S: soluble fraction after cell sonication; P:
pellet fraction after cell sonication. The arrowheads indicate recombinant proteins.
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE

Figure 3 Variation in expression between HE-MBP(Pyr)-fused and His6-MBP-fused proteins.

Expression was induced by 0.5 mM IPTG at 25°C. SDS-PAGE results: A, HE-MBP(Pyr)-Nb (left)
and His6-MBP-Nb (right); B, HE-MBP(Pyr)-PCV2b (left) and His6-MBP-PCV2b (right); C,
EP

HE-MBP(Pyr)-Cago60 (left) and His6-MBP-Cago60 (right); D, HE-MBP(Pyr)-PCV2VHHC3

(left) and His6-MBP-PCV2VHHC3 (right); E, HE-MBP(Pyr)-CdiGMP499 (left) and
His6-MBP-CdiGMP499 (right); F, HE-MBP(Pyr)-CdiGMP026 (left) and His6-MBP-CdiGMP026
C

(right); G, HE-MBP(Pyr)-Prop acid OAS (left) and His6-MBP-Prop acid OAS (right); H,
AC

HE-MBP(Pyr)-FMDV98 (left) and His6-MBP-FMDV98 (right); I, HE-MBP(Pyr)-H5HA10 (left)

and His6-MBP-H5HA10 (right); M, protein molecular-weight marker; C, total cellular protein
level before IPTG induction (negative control); I, total cellular protein level after IPTG induction;
S, soluble fraction after cell sonication; P, pellet fraction after cell sonication. The arrowheads
indicate recombinant proteins.
 ACCEPTED MANUSCRIPT
Table 1. Expression and solubility levels of HE-MBP(Pyr)- and His6-MBP tag-fused proteins.

Protein Expression, % Solubility, %

HE-MBP(pyr) His6-MBP HE-MBP(pyr) His6-MBP

Nb 57.3 56.4 83.0 33.8

PCV2b 45.9 43.2 73.5 62.0

PT
Cago60 61.1 50.0 80.6 52.5

RI
PCV2VHHC3 61.1 59.4 78.6 61.2

CdiGMP499 48.3 42.4 61.5 35.6

SC
CdiGMP026 17.4 0 35.0 0

Prop acid OAS 61.0 58.9 74.7 32.0

FMDV98 64.2

U 52.0 73.8 31.6

AN
H5HA10 49.6 47.2 79.5 57.1

The expression level (%) of fusion protein was calculated based on the density ratio of target
M

fusion protein to the total E. coli expressed-proteins.

The solubility level (%) was calculated based on the density ratio of soluble fusion protein to total
D

fusion protein expressed.

TE
C EP
AC
 ACCEPTED MANUSCRIPT
Table 2. Expression and solubility levels of His6-MBP(Pyr)- and His6-MBP-fused proteins.

Protein Expression, % Solubility, %

His6-MBP(pyr) His6-MBP His6-MBP(pyr) His6-MBP

H5HA10 38.6 47.2 71.7 57.1

HAFnt 46.1 65.7 100 59.4

PT
Cago60 51.6 50.0 71.7 52.5

RI
FMDV98 52.8 52.0 54.3 31.6

Prop acid OAS 47.0 58.9 59.5 32.0

SC
The expression level (%) of fusion protein was calculated based on the density ratio of target
fusion protein to the total E. coli expressed-proteins.

U
The solubility level (%) was calculated based on the density ratio of soluble fusion protein to total
fusion protein expressed.
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Table 3. Expression and solubility levels of HE-MBP(Pyr)- and His6-MBP(Pyr)-fused proteins.

Protein Expression, % Solubility, %

HE-MBP(pyr) His6-MBP(Pyr) HE-MBP(pyr) His6-MBP(Pyr)

Cago60 61.1 51.6 80.6 71.7

PCV2VHHC3 61.1 26.2 78.6 56.4

PT
CdiGMP499 48.3 28.8 61.5 57.3

RI
FMDV98 64.2 52.8 73.8 54.3

H5HA10 49.6 38.6 79.5 71.7

SC
NLSFMD 41.9 22.2 59.3 54.5

Prop acid OAS 61.0 47.0 74.7 59.5

U
The expression level (%) of fusion protein was calculated based on the density ratio of target
AN
fusion protein to the total E. coli expressed-proteins.

The solubility level (%) was calculated based on the density ratio of soluble fusion protein to total
M

fusion protein expressed.

D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Supplementary material

PT
RI
U SC
AN
Figure S1. Variations in expression of between His6-MBP(Pyr)- and His6-MBP-fused proteins.
Expression was induced by 0.5 mM IPTG at 25°C. A: His6-MBP(Pyr)-H5HA10 (left) and
M

His6-MBP-H5HA10 (right). B: His6-MBP(Pyr)-HAFnt (left) and His6-MBP-HAFnt (right). C:

His6-MBP(Pyr)-Cago60 (left) and His6-MBP-Cago60 (right). D: His6-MBP(Pyr)-FMDV98 (left)
D

and His6-MBP-FMDV98 (right). E: His6-MBP(Pyr)-Prop acid OAS (left) and His6-MBP-Prop

acid OAS (right). M: protein molecular-weight marker; C: total cellular protein level before IPTG
TE

induction (negative control); I: total cellular protein level after IPTG induction; S: soluble fraction
after cell sonication; P: pellet fraction after cell sonication. The arrowheads indicate recombinant
proteins.
C EP
AC
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE

Figure S2. Variations in expression between HE-MBP(Pyr)- and His6-MBP(Pyr)-fused proteins.

Expression was induced by 0.5 mM IPTG at 25°C. A: HE-MBP(Pyr)-Cago60 (left) and
His6-MBP(Pyr)-Cago60 (right). B: HE-MBP(Pyr)-PCV2VHHC3 (left) and
EP

His6-MBP(Pyr)-PCV2VHHC3 (right). C: HE-MBP(Pyr)-CdiGMP499 (left) and

His6-MBP(Pyr)-CdiGMP499 (right). D: HE-MBP(Pyr)-FMDV98 (left) and
His6-MBP(Pyr)-FMDV98 (right). E: HE-MBP(Pyr)-H5HA10 (left) and His6-MBP(Pyr)-H5HA10
C

(right). F: HE-MBP(Pyr)-NLSFMD (left) and His6-MBP(Pyr)-NLSFMD (right). G:

AC

HE-MBP(Pyr)-Prop acid OAS (left) and His6-MBP(Pyr)-Prop acid OAS (right). M: protein
molecular-weight marker; C: total cellular protein level before IPTG induction (negative control);
I: total cellular protein level after IPTG induction; S: soluble fraction after cell sonication; P:
pellet fraction after cell sonication. The arrowheads indicate recombinant proteins.
 ACCEPTED MANUSCRIPT

PT
RI
U SC
AN
M
D
TE

Figure S3 SDS-PAGE analysis of HE-tagged protein purification using Ni-NTA resin. A:

EP

HE-MBP(Pyr)-Cago60 (left) and His6-MBP(Pyr)-Cago60 (right), B: HE-MBP(Pyr)-FMDV98

(left) and His6-MBP(Pyr)-FMDV98 (right), C: HE-MBP(Pyr)-H5HA10 (left) and
C

His6-MBP(Pyr)-H5HA10 (right), and D: HE-MBP(Pyr)-Prop acid OAS (left) and

His6-MBP(Pyr)-Prop acid OAS (right) were purified from E. coli by affinity chromatography. M:
AC

protein molecular-weight marker; lane 1: total cellular protein extract following IPTG induction;
lane 2: soluble fraction after cell sonication; lane 3: column flow-through; lane 4: 15 mM
imidazole eluate; lane5: 20 mM imidazole eluate; lane 6: 25 mM imidazole eluate; lane 7: 30 mM
imidazole eluate; lane 8: 40 mM imidazole eluate; lane 9: 50 mM imidazole eluate; lane10: 75
mM imidazole eluate; lane 11: 100 mM imidazole eluate; lane 12: 250 mM imidazole eluate; lane
13: 300 mM imidazole eluate; lane 14: 500 mM imidazole eluate. The arrowheads indicate
recombinant proteins.
 ACCEPTED MANUSCRIPT
1. Maltotriose-binding protein as a new fusion tag increases solubility of target proteins in
E.coli.
2. HE-MBP(Pyr)-fusion tag increases the relative expression and solubility of heterologous
proteins in E. coli
3. HE-MBP(Pyr)-tagged proteins can be purified using immobilized Ni(II) affinity
chromatography.

PT
RI
U SC
AN
M
D
TE
C EP
AC