Báo cáo Y học: Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage-specific protein over-expressed in Escherichia coli pptx

Extra terminal residues have a profound effect on the folding
and solubility of a
Plasmodium falciparum
sexual stage-specific
protein over-expressed in
Escherichia coli
Sushil Prasad Sati
1
, Saurabh Kumar Singh
1
, Nirbhay Kumar
2
and Amit Sharma
1
1
Malaria Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India;
2
Department of Molecular Microbiology and Immunology, Hopkins Malaria Research Institute, The Johns Hopkins University
Bloomberg School of Public Health, Baltimore, Maryland, USA
The presence of extra N- and C- terminal residues can play a
major role in the stability, solubility and yield of recombi-
nant proteins. Pfg27 is a 27K soluble protein that is essential
for sexual development in Plasmodium falciparum. It was
over-expressed using the pMAL-p2 vector as a fusion pro-
tein with the maltose binding protein. Six different constructs
were made and each of the fusion proteins were expressed
and purified. Our results show that the fusion proteins were
labile and only partially soluble in five of the constructs
resulting in very poor yields. Intriguingly, in the sixth con-
struct, the yield of soluble fusion protein with an extended
carboxyl terminus of 17 residues was several fold higher.

Various constructs with either N-terminal or smaller C-ter-
minal extensions failed to produce any soluble fusion pro-
tein. Furthermore, all five constructs produced Pfg27 that
precipitated after protease cleavage from its fusion partner.
The sixth construct, which produced soluble protein in high
yields, also gave highly stable and soluble Pfg27 after clea-
vage of the fusion. These results indicate that extra amino
acid residues at the termini of over-expressed proteins can
have a significant effect on the folding of proteins expressed
in E. coli. Our data suggest the potential for development
of a novel methodology, which will entail construction of
fusion proteins with maltose binding protein as a chaperone
on the N-terminus and a C-terminal Ôsolubilization tagÕ.This
system may allow large-scale production of those proteins
that have a tendency to misfold during expression.
Keywords: expression; fusion protein; precipitation; protein
folding; solubility.
Despite the widespread use of fusion protein-based over-
expression vectors for the production and purification of
proteins in Escherichia coli, the molecular or structural
elements which determine protein stability and solubility for
recombinant proteins are not well understood. The solubi-
lity of non membrane-bound proteins is a complex
biochemical phenomena, and it is generally believed that
properly folded proteins are reasonably soluble in aqueous
solutions. There are many factors that affect protein
solubility, and one such player is the amino acid sequence
variation at the amino (N-) and carboxyl (C-) termini. In a
cellular environment, partially folded or misfolded proteins
are generally prone to aggregate formation, and the cell

machinery gets rid of these aggregates by the combined
action of chaperones and intracellular proteases [1–3].
Several studies have shown that the nature of terminal
residues in proteins (i.e. polar or nonpolar) can play a role in
recognition and subsequent action by cellular proteases
[4,5]. In many cases, polar residues at the carboxyl terminal
are able to prevent recognition by tail-specific proteases
[4–7]. Together, these studies point to a complex and
multifactorial nature of protein folding, and indicate that
phenomena like protein stability or its lability are not
completely understood yet. The molecular and structural
elements which determine protein folding are significant
players in the success or failure of over-expression tech-
niques.
We were interested in producing large amounts of a 27K
cytoplasmic protein (Pfg27) from Plasmodium falciparum
for biochemical and biophysical studies. This protein plays a
crucial role in the sexual development of P. falciparum,and
parasites lacking its gene fail to develop sexually [8]. Initial
efforts to express soluble Pfg27 as a fusion protein with
His-tag in the pRSET vector system (Invitrogen) were
unsuccessful as the over-expressed Pfg27 aggregated after
purification resulting in precipitation. Expression systems
with the maltose binding protein (MBP) have been used
routinely to enhance the solubility and yield of fusion
products. Aside from being an efficient tag for affinity
chromatography, MBP is able to act as a molecular
chaperone to enhance the solubility of fused partners
[9,10]. Therefore, various MBP-Pfg27 fusion constructs
were engineered to study the behavior of the expressed

proteins. These constructs had variations in the sequence
andlengthofextraaminoacidresiduesattheterminiof
Pfg27. All six MBP-Pfg27 constructs produced equivalent
amounts of fusion protein as judged by induction analysis.
Intriguingly, only one construct provided proteins of both
high stability and solubility which could be used in
biochemical and structural studies. Our results indicate that
the critical element in obtaining high quality and quantity of
Correspondence to A. Sharma, Malaria Group, International Centre
for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg,
New Delhi 110067, India. Tel/Fax: +91 11 6711731,
E-mail:
Abbreviations: MBP, maltose binding protein.
(Received 11 July 2002, revised 26 August 2002,
accepted 6 September 2002)
Eur. J. Biochem. 269, 5259–5263 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03237.x
Pfg27 was the presence of a carboxyl terminal extension of
17 residues, which seems to function as a Ôsolubilization tagÕ.
The exact mechanism for this phenomenon where an extra
stretch of residues is able to confer enhanced solubility
properties to a protein is not yet known.
MATERIALS AND METHODS
Oligonucleotides were purchased from Genosys, USA.
Various enzymes, the pMAL-p2 vector and amylose resin
were bought from New England Biolabs (NEB), USA. All
other chemicals were obtained from Sigma-Aldrich Co.,
USA.
Expression constructs
The Pfg27 constructs were PCR amplified from P. falcipa-
rum (3D7 strain) genomic DNA by using the following

primers (the restriction sites are shown in bold and
the respective enzymes in parentheses): Pfg27
A
:5¢-AAA
CTGCAGATGAGTAAGGTACAAAAG-3¢ and 5¢-AAA
AAGCTTAATATTGTTGTGATGTGGTTCATC-3¢ (PstI-
HindIII); Pfg27
B
:5¢-AAACTGCAGATGAGTAAGGTA
CAAAAG-3¢ and 5¢-AAACTGCAGTTAAATATTGTTG
TGATGTGGTTCATC-3¢ (PstI); Pfg27
C
:5¢-AAAAAGC
TTATGAGTAAGGTACAAAAG-3¢ and 5¢-AAAAAGC
TTTTAAATATTGTTGTGATGTGGTTCATC-3¢ (Hin-
dIII); Pfg27
D
:5¢-AAAGAATTCATGAGTAAGGTACA
AAAG-3¢ and 5¢-AAACTGCAGTTAAATATTGTTGT
GATGTGGTTCATC-3¢ (EcoRI-PstI); Pfg27
E
:5¢-AAAC
TGCAGATGAGTAAGGTACAAAAG-3¢ and 5¢-AAAA
GCTTTCACTTCGAATTCCATGGTACCAG-3¢ (PstI-
HindIII); Pfg27
F
:5¢-AAACTGCAGATGAGTAAGGTA
CAAAAG-3¢ and 5¢-AAAAAGCTTTTACGACGTTGT
GTGATGTGGTTCATC-3¢ (PstI-HindIII).
PCR was performed using standard protocols and the

product purified using Qiagen PCR purification kits. DNA
fragments were cloned into the expression vector pMAL-p2
resulting in constructs designated Pfg27
A–F
. All constructs
were verified by DNA sequencing in an ABI 310 automated
sequencer using a forward primer that anneals to the malE
gene upstream of the polylinker region and a reverse primer
that anneals to the lacZ alpha sequence downstream of the
first in-frame stop codon in pMAL vector.
Expression and analysis of recombinant proteins
DNA constructs were transformed into BL21 (B834 DE3)
strain of E. coli by the heat shock method and transform-
ants grown in LB broth in presence of carbenicillin
(50 mgÆmL
)1
) and 0.2% glucose. For protein production,
100 mL of bacterial cultures were induced by 0.3 m
M
IPTG
at D of  0.6 and grown for another two hours. The cultures
were spun down at 6000 g, pellets suspended in lysis buffer
(20 m
M
Tris pH 7.5, 200 m
M
NaCl, 1 m
M
EDTA and
1m

M
phenylmethanesulfonyl fluoride) and subjected to
sonication. The cell suspensions were then centrifuged at
26 000 g for 30 min, and the lysates loaded on pre-
equilibrated amylose columns. The columns were washed
with 12 volumes of lysis buffer and the MBP-Pfg27 proteins
eluted with 10 m
M
maltose. Protein fractions were analyzed
by SDS/PAGE and the fusion proteins cleaved with factor
Xa in lysis buffer containing 10 m
M
CaCl
2
.Incaseof
constructs Pfg27
B–F
, the protease cleavage mixtures were
centrifuged at 15 000 g for 10 min in a microcentrifuge. The
resulting supernatants and pellets were loaded on an SDS/
PAGE gel to check the solubility of MBP and Pfg27 after
cleavage. All protein concentrations were measured by UV
absorption at 280 nm and verified by SDS/PAGE. Stand-
ard protocols were followed for western analysis where
Pfg27 was probed with polyclonal anti-Pfg27 Ig produced in
mice.
RESULTS
Design of the pRSET construct
We first attempted to express recombinant Pfg27-(His)6
fusion protein by taking advantage of a prokaryotic

expression vector, pRSET-C (Invitrogen). The coding
sequence of Pfg27 was PCR amplified using gene specific
primers. The antisense primer lacked the final stop codon
andthePCRproductwasclonedintothePvuII site of
pRSET-C. Subsequent sequence analysis as well as expres-
sion upon induction confirmed production of the fusion
protein. However, this construct produced insoluble protein
which aggregated and precipitated upon purification,
making it unacceptable for either functional or structural
studies.
Design of various MBP + Pfg27 fusion constructs
The different constructs generated to examine the role of
extra amino acid residues at N- and C-termini were as
follows (Fig. 1):
Pfg27
A
: This construct encodes a fusion protein in which
Pfg27 has 12 extra residues at its N-terminal (from the
vector backbone) and 17 extra amino acids at its C-terminal.
The natural end of Pfg27 is …HHNNI but in this construct
Pfg27 ends with …HHNNI + LVPWNSKLGTGR
RFT
TS (the terminal polar residues are underlined). It also
has an unexpected D to N mutation at the seventh residue
position of Pfg27. This construct produces highly soluble
and stable protein due to its 17 residue extension.
Fig. 1. Diagrammatic representation of the seven Pfg27 over-expression
constructs. The arrow shows the factor Xa cleavage site, and wedge
represents the D to N mutation in some constructs. Extra N- and
C-terminal sequences are shown along with the original pRSET vector

construct (Pfg27
HIS
) for reference. The underlined residues indicate the
polar end residues.
5260 S. P. Sati et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Pfg27
B
: This construct has the same 12 extra residues at
the N-terminal as Pfg27
A
, retains the D to N mutation, but
has no C-terminal extension. Any difference observed
between the behavior of fusion proteins Pfg27
A
or Pfg27
B
can be directly attributed to the 17 residue extension in
Pfg27
A
. This construct produced insoluble protein as it
lacks the 17 residue C-terminal extension. In addition, it
clearly shows that the accidental D to N mutation does not
effect protein solubility.
Pfg27
C
: This construct has 15 extra amino acid residues at
the N-terminal, does not have the D to N mutation and also
lacks any C-terminal extension. This construct indicates that
the N-terminal variation has no effect on protein solubility.
Pfg27

D
: This construct does not have extra residues at
either termini and contains no mutation. It is noteworthy
that this construct is designed to express Pfg27 in its native
state without any extra residues. It therefore represents a
typically preferred construct. This is a control construct
designed to make wild-type Pfg27 without extensions or the
D to N mutation.
Pfg27
E
: This construct has extensions at the N- and
C-termini and also the D to N mutation. It is closest to
Pfg27
A
in design but has a truncated C-terminal extension
of only seven amino acids which end in polar residues. This
construct indicates that the length of the C-terminal
extension plays an important role.
Pfg27
F
: This construct has the same N-terminal extension
as Pfg27
A,B,E
but lacks a C-terminal extension. It was
designed to address whether presence of polar residues at
the C-terminal is sufficient to confer increased solubility to
the fusion protein. The end sequence of native Pfg27
(…NNI) was changed to (…TTS). Therefore, the terminal
three residues are identical to the ones in Pfg27
A

.
Stability and solubility of various MBP-Pfg27 proteins
The Pfg27
A–F
construct DNAs were freshly transformed
into E. coli cells, and the transformants grown, induced and
processed under identical conditions (see Materials and
methods). The cell pellets were sonicated and the resulting
lysates were loaded onto pre-equilibrated amylose columns.
Sufficient and equivalent amounts of amylose resin were
used for each construct so that the yield differences were
normalized. All experiments were conducted in 100 mL
cultures and the data presented in Table 1 has been scaled to
represent the yields for one liter cell culture. These
constructs were used to express and purify MBP-Pfg27
A–F
fusion proteins by maltodextrin affinity chromatography
according to manufacturer’s instructions. All constructs
showed equivalent levels of protein induction but the
resulting fusion protein had varying solubilities (Fig. 2,
Table 1). The six constructs produced  200 mgÆL
)1
of
fusion protein. However, the behavior of the proteins varied
once the cells had been lysed for downstream processing and
protein purification. Pfg27
A
produced the highest protein
levels in a stable and soluble form (Table 1). Approximately
33% of the total induced protein could be purified off the

amylose column for Pfg27
A
. In contrast, only 7.5% was
eluted off the amylose resin for Pfg27
B–F
.Further,no
protein was detected in the Pfg27
A
column flow through but
5% of the fusion proteins from Pfg27
B–F
did not bind to
amylose resin and were found in the flow through fractions
(Table 1). The latter observation suggests improper folding
of Pfg27 in the case of Pfg27
B–F
fusions due to which these
interacted poorly with the amylose resin, a behavior noted
earlier [9].
In the MBP fusion system, a factor Xa protease site has
been engineered in the multiple cloning site such that MBP
can be released from the over-expressed fusion protein. In
an attempt to obtain native Pfg27, the six fusion proteins
were incubated with appropriate amounts of the factor Xa
protease for cleavage. All fusion proteins cleaved success-
fully under identical reaction conditions. The stability and
solubility of MBP after factor Xa cleavage was found high
and identical in all cases. However, the resulting Pfg27
proteins from constructs Pfg27
B–F

were labile, and precipi-
tated immediately after cleavage (Fig. 3). The identity of the
precipitated protein (Pfg27) was confirmed by doing western
blot analysis using anti-Pfg27 polyclonal antibodies. In
sharp contrast, Pfg27
A
produced Pfg27 that remained
Table 1. Expression and purification analysis of six MBP + Pfg27
constructs.
Amount of fusion protein (mg)
Construct Flow through
Eluted fusion
protein
Final yield
of Pfg27
Pfg27
A
06624
Pfg27
B
12 13 0
Pfg27
C
10 15 0
Pfg27
D
10 17 0
Pfg27
E
10 14 0

Pfg27
F
13 15 0
Fig. 2. (A–F) Protein expression and purification profile of Pfg27
A–F
constructs by SDS/PAGE analysis. Lane 1, protein standards; lane 2,
uninduced cell pellet; lane 3: induced cell pellet; lane 4, induced cell
supernatant; lane 5, induced cell pellet; lane 6, amylose column flow-
through; lane 7, eluted MBP-Pfg27–70K fusion protein marked by an
arrow.
Ó FEBS 2002 Extra terminal residues affect protein solubility (Eur. J. Biochem. 269) 5261
soluble could be purified to homogeneity, and subsequently
crystallized. The final yield of Pfg27 from Pfg27
A
was
24 mgÆL
)1
of starting culture while it was 0 for Pfg27
B–F
.
Effect of temperature on the expression profiles
We examined the role of temperature in enhancing the
solubility properties of various fusion proteins. For some
constructs, cell cultures were grown at 37 °C but the
temperature dropped to 30 °Cor25°C after induction.
However, no discernible difference in the relative protein
solubility profiles was observed. Indeed, the drastic differ-
ence in fusion protein quality between Pfg27
A
and the rest of

the constructs was retained. Therefore, the differing protein
solubilities were inherent in the constructs and could not be
modulated by varying the ambient growth conditions.
DISCUSSION
The routine production of recombinant proteins in soluble
and biologically active form remains a challenge. In this
context, MBP and other fusion protein expression systems
have been widely used, both because the fusions serve as
efficient purification tags and because they are able to
promote the solubility of the fused partner [9,10]. In the
present study, we engineered several constructs with the
overall aim of obtaining high quality Pfg27 protein for
biochemical and biophysical applications. In the first
instance, we used one of the most commonly used vehicles
for over-expression, namely the pET vector as part of
pRSET system. Although reasonable amounts of Pfg27
were produced upon induction, this construct failed to
produce protein in a soluble form. Subsequently, the MBP
system was used and proved useful in producing at least
partially soluble proteins from constructs Pfg27
A–F
.We
found that fusion to MBP did indeed promote the proper
folding of Pfg27 into a native and stable form but with a
caveat. Our results suggest that the dramatic positive effect
on stability and solubility of Pfg27 produced using the
construct Pfg27
A
can be attributed to the presence of 17
extra residues at the C-terminal end. It is probable that

MBP and the C-terminal extension both contribute to the
high yield and solubility of Pfg27
A
in a complex process of
assisted protein folding.
To dissect further the structural elements responsible for
the enhanced solubility phenomenon observed in Pfg27
A
,
we identified three key issues: (a) the role of the N-terminal
extension; (b) the role of the D to N mutation; and (c) the
role of the sequence and length of the C-terminal extension.
To address whether presence of extra residues at the
N-terminal of Pfg27
A
were responsible for its high yields, we
engineered constructs Pfg27
B
, Pfg27
C
,Pfg27
E
and Pfg27
F
which share similar N-terminal extensions. Clearly, the
presence of these extensions was not enough to yield soluble
fusion protein. Next, we addressed whether the differing
solubilities were due to the accidental D to N mutation in
Pfg27
A

. It is well documented that the solubility of proteins
can be affected severely by single amino acid mutations.
However, in the present study we can exclude this possibility
as the D to N mutation is conserved in four of the six
constructs (Fig. 1), which nonetheless retain the contrasting
solubility profiles for their respective fusion proteins.
Finally, to address whether the presence of terminal polar
resides and the exact length of the C-terminal extension were
responsible for the observed Ôsolubilization effectÕ in Pfg27
A
,
we engineered two constructs Pfg27
E
and Pfg27
F
.Once
again, these constructs yielded only partially soluble fusion
protein.
The postcleavage precipitation of Pfg27 from constructs
Pfg27
B–F
indicated misfolding of Pfg27. It is probable that
these fusions were maintained in solution due to the well
known Ôsolubilization effectsÕ of MBP. Although MBP
remained soluble after cleavage, Pfg27 precipitated. These
experiments also highlight the central issue that solubility of
fusion proteins is not necessarily indicative of either proper
folding or of increased solubility of the protein of interest.
We propose that the success of fusion protein systems
should be ascertained only once the protein of interest has

been cleaved off and shown to retain both its folded state
and its biological activity.
It is possible that the exact sequence and length of the
17 residue extension together contribute Ôsolubilizing and
stabilizing effectsÕ observed in Pfg27
A
. This phenomenon,
where extra C-terminal residues affect the stability of
an over-expressed protein, has some precedence (see
Table 2). A more extensive study can now be undertaken
to verify whether segments like the 17 residue extension at
the C-terminus of Pfg27
A
can be used in a more generic
fashion.
In the postgenomic era of functional genomics, structural
genomics and an expanding scope for biotechnology
products, the production of large amounts of soluble native
protein has necessarily taken the center-stage. Our findings
Fig. 3. SDS/PAGE analysis of Pfg27
B
(A) and Pfg27
A
(B) cleavage
with factor Xa. (A) Lane 1, protein standards; lane 2, cleavage mixture;
lane 3, cleavage mixture supernatant; and lane 4, cleavage mixture
pellet. Proteins Pfg27
C–F
gave identical precipitation profiles. (B) Lane
1, Pfg27

A
fusion protein; and lane 2, cleavage mixture of MBP-Pfg27
A
fusion protein. Protein produced from constructs Pfg27
C–F
showed
identical precipitation behavior.
Table 2. Effect of C-terminal extensions on various proteins expressed in
E. coli.
Protein Variation Effect Reference
Arc repressor C-terminal tail Increased stability [4]
Lambda repressor
protein
C-terminal tail Increased stability [5]
Ara C C-terminal tail Increased stability [11]
Aldehyde
dehydrogenase
C-terminal tail Increased stability [12]
5262 S. P. Sati et al. (Eur. J. Biochem. 269) Ó FEBS 2002
highlight the contribution of extra C-terminal residues in
producing recombinant Pfg27 in a native, folded state which
is now suitable for biochemical, biophysical and immu-
nological characterization. Structural elements like the
17 residue C-terminal extension may have widespread
application in recombinant protein production. More
significantly, this study highlights the complex and
multifactorial nature of protein folding.
ACKNOWLEDGEMENTS
We thank the past and present members of the Malaria Group,
ICGEB, New Delhi for help and discussions. N.K. is supported by the

National Institutes of Health grant AI46760. A.S. is supported by an
International Wellcome Trust Senior Research Fellowship.
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