41NISR Research GRANT
2009 Research Grant

Analysis and Prevention of Inclusion Body in E. coli Overexpression Systems Using Short Peptide Tags
Yutaka KURODA
Institute of Engineering, Tokyo University of Agriculture and Technology

Research aims
Escherichia coli (E. coli) can yield high amount of proteins at a low cost, and it is thus frequently used for the expression of recombinant proteins. However, many recombinant proteins express as inclusion bodies, which are insoluble protein aggregates forming in the interior of E. coli
cells. Protein recovered from inclusion bodies can sometimes be refolded into their native states, but, due to theirdiverse physicochemical properties, a versatile refolding
method is still lacking. Here, we report a systematic analysis of the relationship between in vivo inclusion body formation and in vitro solubility using SEP-tags, which areshort Solubility Enhancing Peptide tags consisting of aglycine and charged amino acids, and which were developed in our laboratory (ref 1, 2; patent pending). In addition, we examine the ability of SEP-tags to prevent inclusion body formation in E. coli.
Methods
We designed three SEP-tags with different lengths (containing 3, 6, and 9 charged amino acid residues), and fusedthem to the C-terminus of 6 model proteins: CAD 1-86
C34S, GLuc (ref 3), N-intein (ref 4), GFP (ref 4), VanX
E181A, and P57. SEP-tags containing arginines were fusedto the basic protein (CAD 1-86 C34S); whereas SEP-tags
containing aspartic acids were fused to the acidic proteins(N-intein, GFP, VanX E181A, GLuc, and P57) (Table 1).
All of the recombinant variants were overexpressed in E.
coli strains BL21 (DE3) pLysS, and JM109 (DE3) pLysS at

37, 30, and 25°C. The effect of SEP-tags on inclusion bodyformation was analyzed by SDS-PAGE and gel band densitometry. Furthermore, we measured the effect of SEP-tagson the activities of GFP, VanX, and GLuc.
Results and Discussions
SEP-tags are essential in this study, which was aimed atrationally analyzing the effects of in vitro protein solubility
on inclusion body formation, because they can modulate invitro protein solubility without (much) affecting their folding structure or stability. Overall, the SEP-tags significantlyreduced the formation of inclusion body. One example isshown in Figure 1 where SEP-tags containing 3, 6, and 9
aspartic acids were attached to N-intein, an acidic proteinwith a molecular weight of 14.4 kDalton. The extent of reduction was directly dependent on the number and type of
charged amino acids contained in the SEP-tags (Figs. 1 and2). These observations confirm that the formation and/or
prevention of inclusion body is intimately related to protein
solubility, and that improvement in solubility can prevent orsignificantly reduce the formation of inclusion body. We
observed very similar effect in both JM109 (DE3) pLysS
and BL21 (DE3) pLysS strains of E. coli.
The types of the charged residues included in the SEPtags strongly influenced its effect on preventing inclusion
body. SEP-tags containing aspartic acid increased the expression of GLuc in the soluble fraction to 80%, however,
the increase resulting from arginine tags were merely 13%.
This result suggests that the compatibility of the physicochemical nature of the target protein with the SEP-tag

Table 1. List of model proteins.

CAD 1-86 C34sN-intein
GLuc
VanX E181A
VanX
GFP
P57MW(kDa)

pI

NoTag

C3D

C6D

C9D

C3R

C6R

C9R

10.0
14.4
18.6
23.6
23.6
27.2
56.7

9.8
4.4
6.7
5.8
5.6
5.8
5.2







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—

—
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—

—
—
—
—


—

—
—
—
—

Variants that were constructed are indicated by a circle (). All of the variants were expressed using a pAED4 expression vectors (ref 57). MW (kDa)and pI indicate, respectively, the molecular weight and the isoelectric point calculated from the untagged (original) sequences. VanX E181A is an inactive
mutant of VanX, which was used because the wild type VanX exhibits a lysis activity when expressed in E coli. The gene encoding the model proteins wereintroduced using an NdeI and an NcoI restriction site. The SEP-tag was located downstream the DNA sequence encoding the model protein.

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Table 2. Relative activity of proteins tagged with SEP-tags relative tothat of non tagged proteins.

Fig. 1. SDS-PAGE of N-intein expressed in BL21(pLysS) at 37°C. M,A, P, and S indicate the molecular weight marker, the whole cell, the precipitate (inclusion body) and the supernatant, respectively. C0D is the untagged protein. C3D, C6D and C9D indicate, respectively, N-intein N
tagged with 3, 6, and 9 aspartic acid residues. The blue line indicates theN-intein band.

SEP-tagged proteins

Relative activity (%)

GFP C9D
VanX C9D
GLuc C9D

6879130

Finally, the activity of SEP-tagged GFP, and VanX dropped
by 32% and 21%, respectively, whereas that of SEP-tagged
GLuc increased by 30% (Table 2). We considered that thediscrepancies were within the limit of our experimental settings and that the tags did not significantly influence thefunction of these proteins.
Conclusions
Here, we have used SEP-tags to show, in a quantitative
manner, that protein solubility is an essential factor determining the formation of inclusion body in E. coli. SEPtags, which can regulate in vitro protein solubility, appears
to be a versatile method for reducing the formation of inclusion body, leading to increased yield of soluble, functional native proteins. As for SEP-tag design asparagines
should be used for acidic proteins and arginines for basicproteins (ref 2, patent pending).
References1.

2.
Fig. 2.

Fraction of protein expressed in the supernatant.

residues (either acidic or basic) is essential in enhancing
protein solubility and thereby reducing the amount of protein expressed as inclusion body. As a practical rule, SEPtags containing arginine should be used for basic proteins
and aspartic acid for acidic proteins.
The expression temperatures are well known to influence
the formation of inclusion body, and we indeed observedsome influence on the effect of the SEP-tags. For example,although, at 37°C, the addition of SEP-tags had essentiallyno effect on the fraction of CAD 1-84 C34S and P57 expressing as inclusion body; at 25°C the fractions of CAD 184 C34S, and P57 in the soluble fraction increased to, respectively, 80% and 44% upon addition of the SEP-tags.
We hypothesize that SEP-tags had no effect at 37°C, because both proteins were unfolded or partly unfolded,
which decreased the protein’s solubility, whereas SEP-tags
were efficient at 25°C where the proteins are stably folded.

3.

4.

5.

6.

7.

A. Kato, K. Maki, T. Ebina, K. Kuwajima, K. Soda, and Y. Kuroda,
Mutational analysis of protein solubility enhancement using short
peptide tags. Biopolymers, 85: 12–18 (2007)
Japanese patent application 2007-303780 A. Kato S. Soya, N. Izumikawa, Y. Kuroda Method for calculating peptide solubilities anddesign and construction of solubilizing peptide tags deposited onNovember 22, 2007. Depositor: TUAT
T. Rathnayaka, M. Tawa, S. Sohya, M. Yohda, and Y. Kuroda, Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli. Biochim Biophys Acta, 1804:1902–1907 (2010)
T. Kamioka, M. Tawa, S. Sohya, T. Yamazaki, and Y. Kuroda, Improved protein splicing reaction for low solubility protein fragments
without insertion of native extein residues (NERs). Biopolymers(Peptide Science), 92: 465–470 (2009)
M. M. Islam, S. Sohya, K. Noguchi, S. Kidokoro, M. Yohda, and Y.
Kuroda, Thermodynamic and structural analysis of highly stabilized
BPTIs by single and double mutations. PROTEINS, 77: 962–970(2009)
M. M. Islam, S. Sohya, K. Noguchi, M. Yohda, and Y. Kuroda, Crystal structure of an extensively simplified variant of bovine pancreatic
trypsin inhibitor in which over one-third of the residues are alanines.
Proc. Natl. Acad. Sci. USA, 105: 15334–15339 (2008)
Y. Kuroda, and P. S. Kim, Folding of bovine pancreatic trypsin inhibitor (BPTI) variants in which almost half the residues are alanine.
J. Mol. Biol., 298: 493–501 (2000)