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Introduction
Pituitary adenylate cyclase activating polypeptide
(PACAP) has a specific receptor, PAC1, and shares 2
receptors, VPAC1 and VPAC2, with vasoactive intestinal
peptide (VIP)[1]. The activation of multiple receptors by
PACAP or VIP has broad physiological effects on nervous,
endocrine, cardiovascular, reproductive, muscular, and
immune systems[2]. Different effects are mediated by different
receptors; for example, VPAC2 activation enhances glucose
disposal by stimulating insulin secretion, while VPAC1
activation elevates hepatic glucose
output[3_6]. It is recognized that a VPAC2-selective agonist can enhance glucose
disposal by stimulating insulin secretion without causing
increased hepatic glucose production, thus VPAC2 selectivity
has been identified as a prerequisite for potential type II
diabetes treatment[4,7].
rMBAY was designed to imitate BAY 55-9837, which is a
VPAC2-selective agonist developed by Bayer Corporation
(USA) as a potential therapy for type II
diabetes[4]. BAY 55-9837, consisting of 31 amino acid residues, is produced by
chemical synthesis that is time-consuming and costly. To
achieve rapid and efficient purification of the recombinant
VPAC2 receptor agonist as potential treatment for type II
diabetes, we fused rMBAY as a target protein to the
N-terminus (Cys1) of a modified intein from Saccharomyces
cerevisiae (Sce VMA intein, 454 amino acids), which in turn
links to the chitin-binding domain
(CBD)[8,9]. The CBD allows the binding of the fusion protein to a chitin column and
the intein is capable of undergoing peptide bond cleavage at
its terminus. The C-terminal residue
(Asn454) of the intein was mutated to an alanine, which blocked the splicing
reaction but still allowed an N-S acyl
rearrangement at the intein N-terminus (Cys1) leading to the formation of a thioester
linkage between the target protein and the intein. Cleavage
of the thioester bond can be induced by thiol reagents, such
as 1,4-dithiothreitol (DTT), β-mercaptoethanol, or cysteine.
The use of DTT or β-mercaptoethanol results in the
formation of a thioester bond between the thiol compound and the
C-terminal residue of the target protein. This thioester is not
stable and hydrolyzes to yield a free C-terminus. As a result,
rMBAY is released from its affinity tag without the use of a
protease and separated from its fusion partner on the same
column.
BAY 55-9837 not only has an extra N-terminal methionine
coded by initiator codon ATG, but also a displacement of
Val17 with Leu17 (Figure 1). The preliminary bioactivity
assay showed that rMBAY had an effect on insulin release and
reducing the plasma glucose after intraperitoneal injection
with high concentrations of glucose to the NIH mice. A
novel recombinant peptide was produced effectively for it
further development as a new treatment to type II diabetes.
Materials and methods
Synthesis of rMBAY gene The rMBAY gene was
designed according to the bias of E coli
for the codons to ensure the high expression in
E coli. The gene was synthesized and amplified in 2 steps using 4 oligonucleotides
primers as described[10](Figure 2).
Four primers for the synthesis of rMBAY were F1:
5'-NNNNNN CATATG CAT AGC GAT GCG GTG TTT ACC GAT AAC TAT ACC CGT CTG CGT AAA CAG -3',
containing a NdeI site (underlined ); F2: 5'-CTG GCG GCG AAA
AAA TAT CTG CAG AGC ATT AAA AAC AAA CGT TAT-3'; F3: 5'-CGC CAG CTG TTT ACG CAG ACG GGT ATA GTT
ATC GGT AAA CAC CGC ATC GCT ATG CA-3'; and F4: 5'-NNNNNN GCTCTTC C GCA ATA ACG TTT GTT TTT AAT
GCT CTG CAG ATA TTT TTT CGC 3', containing a
SapI site (underlined). "N" represented the protecting base.
The PCR product containing the synthesized gene was
purified on a Qiagen QIAquick column (Qiagen, USA). After
double digestion with NdeI and SapI (NEB, USA), the
purified DNA fragment was directly ligated to a
gel-purified NdeI-SapI double digested pKYB vector (NEB) to yield pKY-BAY
(Figure 2). Insertion of the rMBAY gene was verified by
DNA sequencing using the T7 promoter as the sequencing
primer.
Fermentation of engineering strain pKY-BAY-ER2566
The pKY-BAY plasmid was transformed to the E
coli strain ER2566 [fhuA2(lon) ompT lacZ::T7 gene1 galsulA11 D
(mcrC-mrr) 114::IS10 R (mcr-73::miniTn10_TetS) 2R
(zgb-210::Tn10_TetS) endA1 (dcm), NEB], which allowed for
IPTG-regulated expression of the T7 RNA polymerase. The fermentation
was carried on in a CF-5L fermenter (Biotop, Taiwan). Three
hundred mL cultures were grown in an air shaker (250 rpm) at
37 oC in LB medium containing 50 mg/L of kanamycin
overnight, and then inoculated into 3 L LB containing 1%
glycerin in 5 L fermenter. At
A600=8_8.5, IPTG was added to a final concentration of 0.4 mmol/L to induce T7 RNA
polymerase-based expression. The culture was cooled to 30
oC and allowed to continue expression for 4 h. SDS-PAGE was
used to identify the expression of the fusion protein.
Purification of target peptide Five grams of induced
cells (wet weight) were resuspended in 60 mL of column buffer
(20 mmol/L Tris-HCl, 0.5 mol/L NaCl, 1 mmol/L EDTA, pH 8.0)
and then disrupted by pulse sonication. The supernatant
(soluble fraction) was separated from cell debris by
centrifugation at 20 000×g for 30 min and passed through a
column (2.5×10 cm) packed with 20 mL chitin beads at a flow
rate of 0.5 mL/min. After the supernatant were loaded on the
column, the flow rate was raised to 2 mL/min and the column
was thoroughly washed with more than 10 bed volume of
the column buffer until the protein content of the eluate
reached a minimum (A595<0.05 as measured by Bradford
assay). Column buffer (60 mL) containing 100 mmol/L
β-mercaptoethanol was then quickly passed through the
column in order to distribute β-mercaptoethanol evenly
throughout the resin and the column flow was stopped. The
column was incubated at 16 oC for 24 h. Fractions containing
rMBAY were obtained by eluting the column with the
column buffer. Part of the fusion protein eluted with the target
peptide was removed by ultrafiltration (retention molecular
of 50 kDa). Protein concentrations were estimated by the
Bradford method. The rMBAY was further purified by
dialysis overnight at 4 oC to remove β-mercaptoethanol.
SDS-PAGE and Tricine-SDS-PAGE (according to
Yang et al[11]) were both used to identify the purification of the target
peptide. Laser Flying Mass Spectrometry analysis of the
purified peptide was performed in the Test Center at the
Military Physic Academy of Science (Beijing, China).
Cell lines and membrane preparation The cells clones
used were referred to as PAC1-CHO (receptor density of
3.3±0.3 pmol/mg protein), VPAC1-CHO (receptor density of
1.3±0.2 pmol/mg protein), and VPAC2-CHO (receptor
density of 1.1±0.2 pmol/mg protein), as earlier
described[10]. The cells were maintained in DMEM medium supplemented with
10% bovine serum albumin (BSA) and 0.8 mg/mL G418 with
an atmosphere of 95% air, 5% CO2 at 37
oC. Membrane preparation and competition binding assay were performed
following the protocol described by Gourlet et
al[15]. The membranes were prepared from PAC1-CHO, VPAC1-CHO, and
VPAC2-CHO. The cells were harvested with a rubber
policeman and pelleted by low speed centrifugation. The
supernatant was discarded and the cells were lysed in 1 mmol/L
NaHCO3 solution and immediately frozen in liquid nitrogen.
After thawing, the lysate was first centrifuged at 4
oC for 10 min at 400×g, and the supernatant was further centrifuged at
20 000×g for 10 min. The pellet resuspended in 1 mmol/L
NaHCO3 was used immediately as a crude membrane
prepara-tion.
Competition binding assay and cAMP accumulation
assay To measure the receptor binding of rMBAY, 10 µg
membrane was incubated with 0.1
nmol/[125I]PACAP38 (1813.14 Ci/mmol, Phoenix Pharmaceuticals, Mountain View, CA, USA)
in the presence of increasing concentrations of rMBAY
peptide, in a total volume of 100 µL of 20 mmol/L HEPES (pH
7.4), 150 mmol/L NaCl, 0.5% BSA, 2 mmol/L
MgCl2, and 0.1 mg/mL bacitracin. After incubating at 37
oC for 20 min, the bound ligand was collected on GF/C filters pretreated with
0.1% polyethylenimine. The filters were washed with cold
25 mmol/L NaPO4 containing 1% BSA and counted onto a
gamma counter. Nonspecific binding was defined as the
residual binding in the presence of 1 mmol/L RMPACAP (a
recombinant analogue of PACAP38[12]) and was always
below 20% of the total binding. Each assay was performed
at least 3 times.
The PAC-CHO, VPAC1-CHO, and VPAC2-CHO cells cultured in the DMEM nutrient at 37
oC were scraped off with rubber policeman and washed with PBS twice; the density of
the cells was adjusted to 2×106/mL. rMBAY was added to
500 µL cell suspension, and the working concentrations of
the peptide were changed from
1×10-12 mol/L to
1×10-5 mol/L. The reactions were incubated at 37
oC for 5 min and then were incubated at room temperature for 20 min after 2 volume
0.2 mol/L HCl was added. The mixture was dissociated by
pipetting up and down until the suspension was
homo-geneous. The precipitate was removed by centrifugation at
1000×g for 10 min, and the supernatant was collected to the
test tube and assayed for cAMP quantities using the
enzyme immunoassay kit for cyclic AMP (Cayman Chemical
Co, Ann Arbor, MI, USA) following the operating
instruc-tions.
Bioactivity assay of reducing plasma glucose
in vivo Masculine NIH mice (25_30 g) fasted overnight (17 h) and
were randomly divided into groups according to the weight;
each group contained 10 mice. Glucose (1.8 mmol/kg) with
or without peptide was intraperitoneally injected to the NIH
mice. Blood was collected from the tail vein before injection
and at 10 min after the injection. The plasma glucose levels
were determined using OneTouch Ultra Meter (Johnson,
USA) and the blood sera were sent to Dongshan Hospital
(Guangzhou, China) for the plasma insulin assay.
Protein analysis The concentration of proteins in the
clear lysate was determined by the Bradford method using
BSA as a standard. The percentage of the purity of the
target proteins were estimated by analyzing
Tricine-SDS-PAGE gel stained with Coomassie blue using Chemilnager
V5.5 software (USA) for image collecting and analyzing. The
cleavage efficiency and the binding efficiency were
calculated as described[12].
Results
Construction of the recombinant plasmid pKY-BAY
The synthesized human rMBAY gene was inserted into the
plasmid vector pKYB by NdeI and SapI, and the site recognized
by SapI disappeared in the recombinant plasmid pKY-ROM.
The recombinant clone identified by PCR and DNA
sequencing (Figure 3) was transformed into E
coli strain, ER2566, and the engineering strain pKY-BAY-ER2566 was
constru-cted. The sequencing of pKY-BAY is shown in Figure 3.
Fermentation of engineering strain pKY-BAY-ER2566
When A600 reached 8.2, IPTG was added to a final
concentration of 0.4 mmol/L, and the culture was cooled to 30
oC. The maximal expression of the fusion protein reached 21% of the
total bacteria protein at 4 h after inoculation and did not rise
much more for a longer induction time (Figure 4, Figure 5
lanes 6 and 7). Thirty grams of cells (wet weight) was
collected from 1 L culture. Approximately 58% of the total
precursor accumulated as soluble form which reached 16% of
the total soluble proteins in the clarified lysate (Figure 5,
lane 8). The growth curve of the engineering strain
pKY-BAY-ER2566 fermented in 5 L fermenter is shown in Figure 6.
Affinity purification and cleavage of fusion
protein After IPTG induction, the rMBAYintein-CBD fusion precursor
accumulated as a soluble product observed in the clarified
lysate and reached approximately 16% of the total soluble
proteins (Figure 6A, lane 4), while a part of the fusion
precursor accumulated as inclusion bodies existing in the deposit
(Figure 6A, lane 3). After passing the supernatant through
the chitin column, more than 98% of the fusion precursor
was bound to the resin due to extremely high affinity of the
CBD for chitin (Figure 6A, lanes 4 and 5). In addition, since
the CBD could not be eluted from the chitin resin under no
denaturing conditions, extensive washing was performed to
remove most non-specifically bound contaminating proteins
(Figure 6, lanes 6 and 7).
Flushing the column with β-mercaptoethanol triggered
the intein-mediated cleavage reaction, which released
rMBAY, whereas the intein-CBD fusion partner remained
bound to the resin. After the induction of the cleavage
reaction by β-mercaptoethanol at 16
oC for 24 h, the target peptide was released from the intein-CBD tag and was eluted
from the column using the column buffer (Figure 6B, lanes 4
and 5). After ultrafiltration, rMBAY with purity over 95%
was obtained. β-mercaptoethanol was removed by dialysis,
and laser flying mass spectrometry showed the precise
molecular weight of rMBAY with purity over 95% as 3887.03
(Figure 7), which was consistent with the theoretical
molecular weight.
Competition binding assay of rMBAY to PACAP
receptors Competition binding of
[125I]PACAP38 on membranes purified
from CHO cells expressing each of the 3 subtypes of
human PACAP receptors identified rMBAY as a VPAC2-
selective peptide (Figure 8). rMBAY competitively displaced
[125I]PACAP38 from VPAC2, with a half-maximal inhibitory
concentration (IC50) of 53±8 nmol/L, whereas a recombinant
PACAP38 analogue[12], RMPACAP, had an
IC50 of 18±5
nmol/L. The IC50 for rMBAY at human VPAC1 and was over
10 µmol/L, whereas no competition was observed up to 50
µmol/L at PAC1.
PACAP receptor activation by rMBAY The
accumulation of cAMP in human PACAP
receptor-transfected cells was used as an index of the agonist activity. rMBAY was a
potent, full agonist of the human VPAC2, with a
half-maximal stimulatory concentration
(EC50) of 0.8 nmol/L. It was
250-fold less potent at human VPAC1
(EC50 of 200 nmol/L, Figure 9)
and had no activity toward the human PAC1.
In vivo effects of rMBAY on insulin release and glucose
disposal in fasted mice rMBAY (50 ng/kg) dramatically
promoted the insulin release and decreased the level of plasma
glucose after intraperitoneal injection with high
concentrations of glucose (1.8 mmol/kg) in NIH mice using vehicle as
control (Table 1).
Discussion
Few VPAC2 agonists have been discovered or
developed until now. Ro 25-1553 is a synthetic cyclic VIP
derivative with the potency of bronchodilator first identified as a
VPAC2 agonist[13_16]. The hexanoyl-VIP (C6-VIP) exhibits
the high selectivity and potency for VPAC2, but recognizes
different receptor domains from those recognized by
Ro 25-1553[17]. BAY 55-9837 is a potent and highly-selective
agonist for VPAC2 generated through site-directed
mutagenesis based on sequence alignments of PACAP, VIP, and
related analogs. Continuous intravenous or subcutaneous
infusion of BAY 55-9837 reduces the glucose area under the
curve following an intraperitoneal glucose tolerance test.
BAY 55-9837 is considered as a novel therapy for type II
diabetes[4].
The recombinant peptide, rMBAY, designed to imitate
BAY 55-9837, was a potential VPAC2-selective agonist with
corresponding activity of reducing plasma glucose.
Compared with BAY 55-9837, rMBAY had an extra N-terminal
Met and an amino acid residue substitute
(Val17/Leu17). The preliminary bioactivity assay showed that rMBAY enhanced
insulin release and glucose disposal. Although it is reported
that the proper modification of the N-terminus of peptides
such as acylation[16] and
hexanoylation[18] helps to increase its selectivity and potency for VPAC2, it still needs to be
further assayed and determined how the changes in rMBAY
contribute to the selectivity and potency of rMBAY to the
VPAC2 receptor and bioactivity.
A recombinant expression system had been used to
produce recombinant, PACAP_derived, mutant peptide, in which
the target peptide was purified as a fusion to the glutathione
S-transferase and was separated from its purification tag by
the cleavage of Factor Xa[7]. In this paper, we used an
intein-mediated purification with an affinity chitin-binding tag
(IMPACT) system from New England BioLabs (Beverly, MA,
USA.) to achieve rapid purification of a VPAC2-selective
agonist using affinity chromatography, but avoiding
protease treatment and extra purification steps.
The expression of the fusion protein reached 21% of the
total bacteria proteins and 16% of the total soluble proteins
(Table 2). A part of the fusion precursor formed insoluble
inclusion bodies resulting in low recovery after sonication
and centrifugation. The final yield of purified rMBAY was
mainly affected by the efficiency of the intein-mediated
cleavage reaction. It had been demonstrated that the higher
temperature and longer time of the incubation helped to improve
the cleavage efficiency. We at last obtained 53 mg rMBAY
from 1 L induced culture when the column was incubated at
25 oC for 24 h, and the cleavage efficiency was approximately
80% (data not shown). Because the intact fusion precursor
protein and the intein-CBD tag remained bound to the chitin
resin during the intein-mediated cleavage and the target
peptide rMBAY elution. Furthermore, no proteases or extra
protein splitting agents were needed. The rMBAY yielded with
high purity (over 95% was obtained by just one-step
purification in a single column).
An efficient production method for a potential
VPAC2-selective agonist with the activity of decreasing the plasma
glucose was established using intein-mediated purification
with an affinity chitin-binding tag. Our work may pave the
way for the exploitation of the recombinant peptide rMBAY
as a novel therapy for type II diabetes.
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