Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart

Abstract

Background:
Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation.

Results:
Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40-45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification.

Conclusion:
Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

Full text

Srivastavaetal. Microb Cell Fact (2019) 18:215
https://doi.org/10.1186/s12934-019-1267-x
RESEARCH
Enhanced production ofrecombinant
serratiopeptidase inEscherichia coli andits
characterization asapotential biosimilar
tonative biotherapeutic counterpart
Vishal Srivastava, Shivam Mishra and Tapan K. Chaudhuri*
Abstract
Background: Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metal-
loprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and
food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and
sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratio-
peptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and post-
operative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current pro-
duction methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli
was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent
proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation.
Results: Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure
of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the
yield was compromised severely. Optimization of protein expression process parameters such as nutrient composi-
tion, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in ser-
ratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically
inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally
similar serratiopeptidase to the commercial counterpart upon refolding and purification.
Conclusion: Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associ-
ated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of
protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological
activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available
counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.
Keywords: Serratia marcescens, Serratiopeptidase, Escherichia coli, Metalloprotease, Inclusion bodies, Heterologous
protein expression, Recombinant mature version serratiopeptidase (rMSrp)
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Open Access
Microbial Cell Factories
*Correspondence: tkchaudhuri@bioschool.iitd.ac.in;
tapanchaudhuri@hotmail.com
Kusuma School of Biological Sciences, Indian Institute of Technology
Delhi, Hauz Khas, New Delhi 110016, India
Background
Proteases are one of the most abundant protein family
represented by those protein molecules which hydro-
lyze substrate protein molecules by disruption of the
peptide bonds in between constituent amino acids [1].
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Srivastavaetal. Microb Cell Fact (2019) 18:215
Extracellular microbial proteases are pivotal for the
growth and survival of protease producing microrgan-
isms [2] as they do protein catabolism in the surrounding
environment producing smaller peptides or amino acids
to fulfill the nutritional requirements of source organ-
ism [3]. Proteases act as virulence factors and are critical
for initiating as well as establishing microbial infections.
Apart from carrying out crucial biological functions,
proteases are equally relevant in a variety of commercial
and industrial applications such as—an additive in deter-
gents, in food processing (meat tenderization, milk coag-
ulation), brewing, leather tanning and paper industry
[4–6]. e hydrolytic activity of proteases is equally rel-
evant for therapeutic purposes. ey are used as an oral
digestive aid, local clearing agents for solubilizing protein
deposits, minimizing inflammation, or as a thrombolytic
agent in thromboembolic disorders [7]. Food and Drug
Administration (FDA) approved at least 12 proteases as a
drug for treating disorders like—hemophilia, stroke, AMI
(acute myocardial infarction), unwanted inflammatory
response, and digestive disorders [8–11]. Such a wide
variety of industrial and therapeutic applications account
for around 60% of worldwide enzyme sales attributed
alone to proteolytic enzymes [12].
Serratia marcescens, a Gram-negative opportunistic
pathogen secretes at least four different types of pro-
teases. e majority of the proteolytic activity exhibited
in the extracellular secretion was attributed to a 50kDa
zinc metalloprotease known as serralysin, serrapeptase,
or serratiopeptidase [13–15]. Serratiopeptidase shows
multidomain architecture containing a zinc atom in
its catalytic site located in the N-terminal domain. e
C-terminal domain of the protein consists of repeat-in
toxin (RTX) glycine-aspartate rich motifs responsible for
the binding of seven calcium atoms to the protein [16].
Broad specificity of serratiopeptidase is essential for the
protein as a virulence factor to exhibit cytotoxicity and
immunomodulation in a variety of hosts [17–21]. e
broad specificity of serratiopeptidase is equally important
for industrial applications such as—a detergent additive,
in food processing, brewing, leather, and paper industry
[22, 23]. Serratiopeptidase shows the potent anti-inflam-
matory and analgesic activity of therapeutic relevance
and sold in the market either as a single component or
as fixed-dose combination (FDC) enteric-coated tab-
lets. e drug is prescribed for treating disorders like—
chronic sinusitis, post-traumatic swelling, fibrocystic
breast disease, bronchitis, healing after molar extrac-
tion, and post-surgical inflammation in several Asian and
European countries [24]. e anti-inflammatory action of
serratiopeptidase is attributed to its ability to break down
insoluble protein exudates, facilitating drainage, and
hydrolyzing inflammatory protein molecules [25]. e
analgesic action of serratiopeptidase possibly functions
through inhibiting the release of pain-inducing amines
[26]. Serratiopeptidase is a potent anti-biofilm molecule
and also disrupts amyloid fibrils invitro as well as invivo
[27, 28].
e industrial and pharmaceutical demand of serratio-
peptidase is fulfilled through growing wild and mutant
strains of S. marcescens in nutrient-rich growth medium
and further extracting the protein out from the extracel-
lular broth. e present approach of production is source
organism dependent and provides a narrow scope of
optimization, hence also limiting the yield [22]. e path-
ogenic nature of the source organism and its association
with a variety of infection ensures the need for an alter-
native approach for serratiopeptidase production. Serra-
tia marcescens associated infections include but are not
limited to ventilator-associated pneumonia, endocarditis,
bacteremia, post-surgical infections, microbial keratitis,
urinary tract infection, meningitis and necrotizing fas-
ciitis [13, 17, 18, 29, 30]. Multi-drug resistant strains of
S. marcescens are associated with clinical outbreaks in
intensive and neonatal care unit are in the high priority
list of World health organization (WHO) for develop-
ing novel antimicrobial therapies [31, 32]. Bulk release of
bacterial biomass is a common thing during large-scale
production of serratiopeptidase and potentially hazard-
ous for associated people with industrial operations.
Recombinant expression of serratiopeptidase in E. coli
based system seems to be a viable solution that will not
only limit the use of native pathogenic source strain but
also provide an opportunity of various expression param-
eters. Optimization of expression parameters would
result in enhancement in yield and even might prove
cost-effective.
Escherichia coli cells are well studied, and a variety of
engineered expression strains of E. coli are available. It
also has a considerably fast growth rate and fermentation
batch turnaround number equal to 300 peryear, which
is farhigher than any of the host systems available [33].
E. coli are nutritionally versatile and in combination with
the above-mentioned properties fit most suitable sys-
tem for heterologous protein expression. E. coli based
expression systems are used for recombinant production
of around 30% FDA approved therapeutically relevant
protein molecules; viz—human insulin, plasminogen
activator, growth hormone [34]. Even after having such
versatility, and Serratia protease genes cloned nearly
30years ago [35] industries prefer the wild source organ-
isms over E. coli based expression. e answer lies in the
fact that E. coli based heterologous expression of pro-
teases causes critical cellular stress due to the associated
catalytic activity of proteases and failure of the expres-
sion system [36]. Sign of failure of expression system is
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Srivastavaetal. Microb Cell Fact (2019) 18:215
often visualized in the form of cell lysis, growth inhibi-
tion, instability of the expression plasmids, lack of protein
expression, degraded protein expression, or deposition of
the proteins into non-functional misfolded aggregates;
i.e., inclusion bodies [37].
e present work demonstrates the successful execu-
tion of an E. coli based alternative method for serratio-
peptidase production in its propeptide devoid mature
form. e expression was carried out in E. coli BL21
C43(DE3) cells designed explicitly for membrane and
toxic protein expression. A significant enhancement in
protein expression was achieved through the optimiza-
tion of expression parameters such as growth medium,
induction point, inducer concentration, temperature,
and duration of induction. e protein expresses in the
form of insoluble non-functional inclusion bodies, which
were further refolded and purified into its function-
ally active folded form. e protein shows the activity,
nature, and conformational features very similar to the
commercially available native version of the protein. e
molecule could be a recombinant biosimilar of serratio-
peptidase for therapeutic purposes and industry-relevant
applications.
Results
Recombinant cloning anddevelopment ofmature
serratiopeptidase expression construct (pMSrp)
Formation of transparent halo around the point inocu-
lated culture (~ 1×106 CFU) of the bacteria was attrib-
uted to the presence of extracellular proteases (shown
in Additional file 1: Figure S1a panel-ii). ere was a
prominent protein band visible around 50kDa molecular
weight in SDS-PAGE gel, lane loaded with extracellular
supernatant from 48h grown bacterial culture (shown in
Additional file1: FigureS1b). Peptide mass fingerprinting
of the corresponding protein band after trypsin digestion
showed 51% sequence coverage with serralysin protease,
also known as serratiopeptidase (shown in Additional
file1: FigureS2).
PCR amplified gene-specific to mature serratiopepti-
dase was 1416bp in size and ligated downstream to T7
promoter in pET23b(+) expression vector in between
NdeI and XhoI restriction sites. e developed recom-
binant plasmid was termed pMSrp. Single digestion of
recombinant plasmid pMSrp generated linearized vec-
tor pMSrp-SD and confirmed the size of the recombi-
nant construct equivalent to 5081bp (Fig.1b MSrp-SD)
while the double digestion with NdeI and XhoI resulted
in two linear fragments of around 3600bp and 1416bp
equivalent to the vector backbone and the serratio-
peptidase (srp) gene insert respectively (Fig. 1b Msrp-
DD). Sequencing results along with restriction digestion
results confirmed the successful cloning of the gene in
pET23b(+) vector and development of recombinant
expression vector pMSrp.
pMSrp expression seems toxic forE. coli cells, andonlyE.
coli C43(DE3) cells expressed theprotein correctly
e number of transformed cells in the presence and
absence of serratiopeptidase gene could explain the
toxicity of the gene. e number of transformants in E.
coli DH5-α after transformation with pET23b(+) (no
gene) and pMSrp (mature gene) were almost equal but
were significantly different in other DE3 variants of E.
coli, suggesting that the presence of gene had some del-
eterious effect on cells. Transformation of the plasmid
pMsrp was not possible in E. coli BL21(DE3)cells despite
repeated attempts, as shown in Fig. 2a. While in other
DE3 variants, viz—C43(DE3), pLysS, and Rosetta(DE3)-
pLysS (RDP) number of successful transformants were
significantly lesser than the vector alone (Fig.2a).
When the protein expression was analyzed in different
DE3 expression strains, the difference in protein expres-
sion was evident. Even after successful transformation,
no visible protein expression was seen in E. coli BL21
(DE3)-pLysS cells (pLysS) as visualized on SDS PAGE
(Fig.2b induced pLysS lane). E. coli Rosetta (DE3)-pLysS
cells (RDP) overexpressed a protein lesser than the actual
size of mature serratiopeptidase, i.e., 50 kDa (Fig. 2b
induced RDP-1 lane). Delayed induction of RDP cells; i.e.,
after OD600 > 1.0 resulted in the expression of the correct
size protein representing ~ 5% of total protein expres-
sion (Fig. 2b induced RDP-2 lane) in comparison to
degraded overexpressed protein which was around 21%
of total protein expression in the cell lysate. e maxi-
mal transformation was found in E. coli C43(DE3) cells
(Fig. 2a pMSrp in C43). e cells were expressing the
protein at correct size and were around 12% of the total
protein (Fig.2b induced C43 lane). Fractionation assay
results shown in Fig. 2c confirmed the overexpressed
protein corresponding to the mature serratiopeptidase
expressed in the form of insoluble inclusion bodies and
goes entirely in the pellet fraction,
Optimization ofexpression parameters resulted
inve‑time more expression ofmature serratiopeptidase
inE. coli cells
Without optimization, mature serratiopeptidase over-
expression constituted about 12% of total protein
expression in the form of insoluble inclusion bodies.
Optimization of various physicochemical parameters
was carried out with a hope of enhanced expression of
recombinant protein. e point of induction was taken
as first parameter for optimization, and this optimization
alone enhanced the total cellular expression of mature
serratiopeptidase by 1.46 times, the expression obtained
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Srivastavaetal. Microb Cell Fact (2019) 18:215
at optical density ~ 0.6–0.8. e total protein expression
was 25.9% at the optical density of 0.5–0.6 in comparison
to 17.7% at 0.6–0.8 optical density measured at 600nm
(Fig.3a, Sections1 and 2).
Effect of nutrient medium composition on mature ser-
ratiopeptidase overexpression was measured by analyzing
the expression in three complex nutrient media compo-
sitions viz—Luria broth, 2YT broth, and Terrific broth.
2YT broth showed the maximum expression, comprising
44% of the total expression. e addition of glucose at 1%
(w/v) negatively affected the level of protein expression,
irrespective of the growth media composition (Fig.3b,
Sections1 and 2). e next parameter chosen for an opti-
mization was inducer concentration. RNA polymerase
found in T7-promoter based vectors is lactose inducible.
IPTG, a synthetic structural analog of lactose is preferred
over lactose since it cannot be metabolized, so the con-
centration of inducer remains constant throughout the
induction. e overexpression of mature serratiopepti-
dase varied from 25 to 44% at different concentrations
of inducer ranging between 0.1 and 2mM (Fig.4a, Sec-
tion1). e maximum recombinant protein expression
was observed at 0.8mM, which was 45.4 ± 1.76% of total
protein expressed (Fig. 4a, Section 2). Change in tem-
perature does not have any significant effect on protein
expression or solubility. Less expression of mature serra-
tiopeptidase was visible at 30°C in comparison to 37°C.
Induction at 25°C or 18°C shown no visible expression
of recombinant protein when observed on SDS-PAGE, as
evident in Fig.4b, Section1. e optimal temperature for
serratiopeptidase expression was 37°C, where expressed
mature serratiopeptidase constituted about 45% of total
protein expression. Post-induction duration for maxi-
mal expression was optimized between 2 and 14h. e
maximal expression of recombinant mature serratio-
peptidase was found after 6 h of induction contribut-
ing 57.9 ± 0.73% of total intracellular protein expression
(Fig.4b, Section2).
Kbp
ab
Marker
C-PCR
M-PCR
1
2
3
4
6
10
pMSrp
pMSrp-SD
pMSrp-DD
Linear pMSrp (~5081bp)
pET23b(+) backbone (~3.6kb)
msrp gene (~1416 bp)a
Marker
Kbp
1
2
3
4
6
10
Fig. 1 Recombinant cloning and development of mature serratiopeptidase specific expression construct. a Representative Agarose gel (1.2%)
showing amplification of ~ 1500 bp gene fragment, particular to the size of mature serratiopeptidase gene (M-PCR). The gene cloned in the
pET23b(+) vector having Ampr for selection. When digested with single restriction enzyme; i.e. NdeI and two different enzymes; i.e., NdeI and
XhoI. b Representative agarose gel (1.2%) shows a linear fragment (pMSrp-SD) of ~ 5000 bp and two fragments equal to the size of plasmid
backbone ~ 3600 bp and insert gene (MSrp) ~ 1500 bp in Lane (pMSrp-DD) respectively confirming the successful insertion/ligation of gene and
construction of recombinant plasmid
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Srivastavaetal. Microb Cell Fact (2019) 18:215
The presence ofplasmid pMSrp slowed downthegrowth
rate ofE. coli cells
Bacterial cells transformed with plasmid-irrespective of
its type or gene insert show differences in growth rate in
comparison to non-transformed cells. ese differences
are resultant of altered internal energetics of the bacte-
rial cells and affect the production rate of recombinant
proteins. e growth kinetics of E. coli C43(DE3) cells
was observed in the presence and absence of mature ser-
ratiopeptidase specific gene under uninduced as well as
50
21% 5%
50
Supernaten
t
Pellet
Induced
Uninduced
Marker
KDa
180
130
70
55
48
35
25
15
12%
Marker
Marker
Marker
Uninduced-C43
Induced-C43
Induced-pLysS
Uninduced-pLysS
Induced-RDP-1
Induced-RDP-2
Uninduced-RDP-1
Uninduced-RDP-2
ac
b
Fig. 2 Selection of E. coli expression host system and expression of mature serratiopeptidase. a Bar graph showing the number of pMSrp plasmid
transformants in different E. coli expression host systems. DH5-α, BL21-DE3, C43, pLysS and RDP represents the E. coli cloning and expression strains
E. coli DH5-α, E. coli BL21 (DE3), E. coli C43(DE3), E. coli BL21 (DE3)-pLysS, and E. coli Rosetta (DE3)-pLysS respectively. b Representative SDS-PAGE gel lanes
are showing expression of mature serratiopeptidase gene product in different E. coli expression systems. While E. coli C43(DE3) cells express protein
at the correct size, in other systems, there is either no visible expression (Induced-pLysS) or degraded expression (Induced-RDP-1) unless induced at
optical density > 1.0 at 600 nm (induced-RDP-2). The percentage contribution of the mature serratiopeptidase in total expressed protein either at
correct molecular weight or in the degraded form is mentioned below the gel lanes. c Representative 12% SDS-PAGE gel showing total cell lysate of
uninduced and induced cell fractions of E. coli C43(DE3) cells showing overexpression of protein equivalent to 50 KDa. Fractionated samples of cell
lysate loaded on SDS-PAGE shows the mature serratiopeptidase expresses in the form of insoluble inclusion bodies and completely goes into the
insoluble fraction; i.e., pellet
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Srivastavaetal. Microb Cell Fact (2019) 18:215
induced conditions. e E. coli C43(DE3) cells without
any presence of plasmid as well as inducer were taken as
a negative control. e obtained growth curve of E. coli
C43(DE3) cells for 12-h duration, as shown in Fig.5, was
used for measuring the specific growth rate of the bac-
terium in different conditions. All the transformed cells
showed a decrease in growth rate as compared to the
wild-type E. coli C43(DE3) cells. It was also evident that
the presence of IPTG as inducer significantly decreased
the growth rate. e calculated specific growth rate of E.
coli C43-de3 cells in each condition are summarized in
Table1.
Refolding andpurication ofisolated inclusion
bodies provided homogenous enzymatically active
serratiopeptidase
125–135 mg serratiopeptidase inclusion bodies were
obtained from a liter of grown culture, which was about
55% pure. Only the washing of inclusion bodies pro-
vided a 10% enhancement in the purity level, giving 100–
120mg of 60–65% pure inclusion bodies (Fig.6a). e
refolding efficiency of the protein was around 50%, and
55–60mg refolded and enzymatically active recombinant
mature serratiopeptidase was obtained after the rapid
dilution (1:100) and concentration. e protein prepared
through refolding has purity around 85–90% meas-
ured through SDS-PAGE (Fig.6b). e trace amount of
denaturants and protein contaminants were removed
through size-exclusion chromatography, providing a
yield of 45–50mg > 98% pure functionally active recom-
binant mature serratiopeptidase (Fig.6c). e yield meas-
urement of enzymatically active refolded recombinant
mature serratiopeptidase from 1l of bacterial culture as
measured by Bradford assay, densitometric analysis, and
activity assay are given below in Table2.
Azocasein based proteolytic assay suggests the specific
activity of purified refolded recombinant mature serra-
tiopeptidase was 1750 ± 5 EU/mg in comparison to com-
mercial standard showing ~ 1820 ± 5 EU/mg (Fig.6d).
Recombinant version mature serratiopeptidase could
be apotential biosimilar tothenative counterpart
oftheprotein
Native-PAGE, along with analytical HPLC results, con-
cluded purified recombinant mature serratiopeptidase
was homogenous preparation of functionally active
monomeric molecules (Fig. 7a, b). e elution peak of
Marker
Uninduced
>0-0.1
>0.1-0.2
>0.2-0.3
>0.3-0.4
>0.4-0.5
>0.5-0.6
>0.6-0.8
>0.8-1.5
66.2KDa
50.0KDa
35.0KDa
Marker
LB-Uninduced
LB-Induced
LB+Glu-Uninduced
LB+Glu-Induced
1-
10
0
20
30
40
50
LB
LB+glu
2YT
2YT+glu TB+glu
TB
>0-0.1
>0.1-0.2
>0.2-0.3
>0.3-0.4
>0.4-0.5
>0.5-0.6
>0.6-0.8
>0.8-1.5
noisserpxenietorP
)llecninietorpdesserpxelatotfo%(
2-
2YT-Uninduced
2YT-Induced
2YT+Glu-Uninduced
2YT+Glu-Induced
TB-Uninduced
TB-Induced
TB+Glu-Uninduced
TB+Glu-Induced
66.2KDa
50.0KDa
1-
noisserpxenietorP
)llecninietorpdesserpxelatotfo%(
35.0KDa
Marker
5
0
30
25
20
15
10
2-
ab
Fig. 3 Optimization of serratiopeptidase expression in E. coli C43(DE3) cells. a Effect of induction on overexpression of mature serratiopeptidase
in E. coli C43(DE3) cells at different time points of growth was observed by supplementing culture with 1 mM IPTG. Section 1 shows the SDS-PAGE
gel loaded with induced samples at different time points. Section 2 shows the bar graph plot representing the relative expression percentage of
mature serratiopeptidase as the average of three independent densitometric analysis. Section 1 of part b shows the overexpression profile of the
recombinant version of mature serratiopeptidase in different growth media on 12% SDS-PAGE gel. Obtained percent values of serratiopeptidase
expression in terms of total intracellular protein expression was averaged out for three independent densitometric analysis and used for plotting
the bar graph shown in Section-2 of part-b
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Srivastavaetal. Microb Cell Fact (2019) 18:215
recombinant preparation coincided with the commercial
version standard eluting at 7.5ml.
In-gel trypsin digestion and peptide mass fingerprint-
ing analysis of generated peptide fragments matched
with S. marcescens serratiopeptidase protein having a
sequence coverage of 53% (shown in Additional file 1:
FigureS3). Circular dichroism (CD) spectra, as well as
intrinsic fluorescence spectra of recombinantly prepared
mature serratiopeptidase, were very similar to the com-
mercially available serratiopeptidase (Fig. 7c). Similarly,
the intrinsic fluorescence emission maxima were 339
and 338 nm for mature recombinant version and the
commercial standard respectively and were identical, as
shown in (Fig.7d).
Discussion
Considering the therapeutical and industrial impor-
tance of serratiopeptidase [22, 24], in the present work,
we focussed on the development of a recombinant DNA
based methodology for the production and purification
of serratiopeptidase in E. coli system. e prominent pro-
tein band around 50kDa in the extracellular secretion of
S. marcescens mtcc7298 strain was the major extracellular
protease of the bacteria called serratiopeptidase [15, 38].
e developed recombinant expression construct pMSrp
contains the gene-specific to mature serratiopeptidase
gene lacking the N-terminal pro-peptide encoding nucle-
otides [35] located downstream to T7 promoter and can
be transcribed in E. coli expression strains encoding T7
RNA polymerase.
When the gene was transformed into different E. coli
expression systems, differences in the number of trans-
formants was evident, which could be correlated to the
toxicity of the gene. E. coli BL21 (DE3) cells repeatedly
failed during transformation, and not a single colony
was formed on the plate. E. coli C43(DE3) cells designed
by Miraux and Walker contains two mutations in −10
region of lacUV5, which allows better overexpression
of membrane proteins. e strain also provides stability
to plasmids encoding toxic genes and allows the expres-
sion of recombinant proteins seems to be toxic in other
E. coli DE3 variants [39]. When pMSrp transformed in
C43(DE3) cells, not only the maximum number of trans-
formants was formed in E. coli C43(DE3) cells but also
the transformed cells expressed serratiopeptidase protein
efficiently.
66.2KDa
50.0KDa
35.0KDa
.1 .2 .3 .4 .5 .6 .7 .8 .9 1.0
Marker
noisserpxenietorP
(
llecninietorpdesserpxelatotfo%
)
IPTG concentration
(in milimolar)
0
20
10
30
40
50
60
1-
2-
Marker
Uninduced
Induced (37
o
C)
Mature Srp
66.2KDa
50.0KDa
35.0KDa
1-
2-
2h 4h 6h 8h 10h 12h 14h
M UI 2h 4h 6h 8h 10h 12h 14h
66.2KDa
50.0KDa
35.0KDa
Post Induction Duration
noisserpxenietorP
)llecninietorpdesserpxelatotfo%(
0
20
10
30
40
50
60
70
Uninduced
Marker
Uninduced
1.11.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
.1 .2 .3 .4 .5 .6 .7 .8 .9 1.01.11.21.3 1.41.51.61.71.81.9 2.0
Induced (30
o
C)
Induced (25
o
C)
Induced (18
o
C)
a b
Fig. 4 Effect of Inducer (IPTG) concentration, temperature, and post-induction duration on serratiopeptidase expression in E. coli C43(DE3) cells.
a Representative 12% SDS-PAGE (section-1) gel showing the effect of inducer concentration on recombinant serratiopeptidase expression at
different concentrations of inducer ranging from 0.1 to 2.0 mM. Section 2 of the image shows the plotted bar graph of average protein expression
at different inducer concentrations obtained from the densitometric analysis of three independent SDS-PAGE gels. b Section 1 represents a 12%
SDS-PAGE gel showing the effect of temperature on mature version serratiopeptidase under uninduced condition (uninduced lane) while other
lanes represent induced cell lysates at different temperatures viz—37 °C, 30 °C, 25 °C, and 18 °C. Section 2 is the plotted bar graph showing
the effect of post-induction duration on the expression of mature recombinant serratiopeptidase. The expression was measured from three
independent sets of experiments and represented in the form of the average value of them, showing the percentage expression of mature
serratiopeptidase in terms of total intracellular protein expression visualized on the SDS-PAGE gel
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Page 8 of 15
Srivastavaetal. Microb Cell Fact (2019) 18:215
Although a good number of transformants were pos-
sible in other E. coli BL21 (DE3) variants, viz—E. coli
BL21 (DE3)-pLysS, E.coli Rosetta (DE3)-pLysS, they all
failed at the protein expression level which could be
attributed to protease associated cytotoxicity [39, 40].
e correct and maximum serratiopeptidase expression
was found in E. coli C43(DE3) cells, which was ~ 12%
of total protein expression resulting in purification of
around 2–2.5mg functionally active serratiopeptidase
from 1-l of culture.
Recombinant version mature serratiopeptidase
exclusively formed insoluble aggregates, i.e., inclusion
bodies. Optimization of overexpression process param-
eters such as induction point, temperature, inducer
concentration., results in enhancement of correct fold-
ing hence solubility of the protein. It also aids in the
enhancement of the yield of the recombinant protein
of interest [41]. Considering these two factors, viz—
solubility and yield, optimization of various process
parameters was carried out, which resulted in 5 times
more expression of the protein providing around 20
times more yield in the form of 40–45mg functionally
active pure mature serratiopeptidase from 1-l of cul-
ture. No effect of process parameter optimization was
seen on the solubility of the protein. Temperature opti-
mization further confirmed the toxicity of the protein
since no visible expression of the protein was found at
lower temperatures, i.e., 25 and 18°C. Failure in achiev-
ing the soluble protein and expression of the protein
in the form of inclusion bodies could be attributed to
pH, osmolarity, redox potential, cofactors, and fold-
ing differences in the intracellular microenvironment
since the protein is an extracellular protein [33, 42].
Usually, when the expression of recombinant protein
goes beyond 2% of the total cellular protein, it results
in unregulated accumulation of the protein insoluble
aggregates known as inclusion bodies [43]. Inclusion
Optical Density (at 600nm)
Time (hours)
Fig. 5 Effect of inducer and different vector constructs on growth kinetics of E. coli C43(DE3) cells
Table 1 Specic growth rate constants (µ) forthe growth
ofE. coli C43(DE3) cells underdierent conditions at37°C
Conditions Specic growth
rate (µ) (h−1)
E. coli C43(DE3) cells 0.5604
E. coli C43(DE3) + IPTG 0.6044
E. coli C43(DE3)-pET23b 0.5464
E. coli C43(DE3)-pET23b + IPTG 0.6091
E. coli C43(DE3)-pMSrp 0.5965
E. coli C43(DE3)-pMSrp + IPTG 0.7091
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Srivastavaetal. Microb Cell Fact (2019) 18:215
Marker
Inclusion body
KDa
ab
cd
116
66.2
50
35
25
18.4
14.4
KDa
116
66.2
50
35
25
18.4
14.4
KDa
116
66.2
50
35
25
18.4
14.4
Marker
Refolded Srp
Flow through
Concentrated Srp
StandardrMSrp
-
(EU.mgActivity ¹)
Marker
E1 E2 E3 E4
Fig. 6 Purification of recombinant mature serratiopeptidase and proteolytic activity assay. Representative SDS-PAGE gels showing a isolated
inclusion body (IB) of recombinant version mature serratiopeptidase from 6 h grown induced culture of E. coli C43(DE3) cells harbouring the
expression plasmid pMSrp, b refolded serratiopeptidase by rapid dilution in ice-cold refolding buffer (refolded srp lane) and protein profile after
concentration (concentrated srp). The lane Flow through was loaded with filtrate collected during concentration using 30 kDa molecular weight
cut-off during concentration. c Representative SDS-PAGE gel showing collected elution fractions (E1–E4) of purified refolded mature version
recombinant serratiopeptidase by size exclusion chromatography. d Protease activity of purified mature recombinant serratiopeptidase (rMSrp) its
commercially available wild counterpart (standard) was measured using azocasein as substrate. The obtained specific activity of each one is plotted
in the form of bar graph with error bars representing the standard error calculated from three independent experiments
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Srivastavaetal. Microb Cell Fact (2019) 18:215
bodies formed during heterologous overexpression
constitute 50–60% of the recombinant protein of inter-
est, having the presence of fewer protein impurities [44]
and could serve a better source of protein preparation
if the proper method of their solubilization and refold-
ing could be devised. A variety of proteins are success-
fully recovered from accumulated inclusion bodies,
which are conformationally and functionally, similar to
the native protein [45]. Inclusion bodies representing
mature serratiopeptidase were isolated from E. coli
C43(DE3) cells and refolded in ice-cold refolding buffer
with an efficiency of around 50%. e purified mature
serratiopeptidase from refolded inclusion bodies was
identical to the commercially available native version
on the molecular level (monomeric) as well as func-
tional level (activity assay). Comparison of the second-
ary and tertiary structure of recombinant one with
the commercial one through circular dichroism and
intrinsic fluorescence emission suggests both are very
similar. e specific activity of recombinant version is
comparable to the native version, and it could serve as
a recombinant biosimilar for a variety of biotechnologi-
cal and industrial applications.
Conclusions
It is nearly impossible to express proteases in E. coli as a
functional protein due to their associated catalytic activ-
ity. Unregulated intracellular expression of proteases
often results in cell death, hindrance in growth, lack of
expression, degraded expression, or expression of the
Table 2 Protein yield comparison at dierent steps
of purication of the recombinant version of mature
serratiopeptidase
Step Yield (mg) Purity (%) Activity (EU/mg)
Isolation 130 ± 5 55 Not measured
Washing 110 ± 10 60–65 Not measured
Refolding by dialysis 57.5 ± 2.5 85–90 1650 ± 5 EU/mg
Gel filtration 47.5 ± 2.5 > 98 1750 ± 5 EU/mg
SP1
a
b
d
c
SP2 M1 M2
1μg 5μg 5μg 1μg
Bovine serum albumin (66.4kDa)
)UAm(mn082VU
Elution (minutes)
Bovine serum albumin
Lysozyme
Commercial Serratiopeptidase
Recombinant serratiopeptidase
serratiopeptidase (50.2kDa)
wavelength(nm)
wavelength(nm)
01xΘ 6)1-lomd.2mc·ged(
).U.A(ytisnetnI
MSrp
Standard
MSrp
Standard
Fig. 7 Biophysical characterization of recombinant mature version serratiopeptidase. a Native PAGE loaded with 1 μg and 5 μg of recombinant
mature version serratiopeptidase (SP1, SP2) and bovine serum albumin (BSA) as a marker (M1, M2) suggests the purified protein is monomeric. b
Analytical HPLC of the recombinant mature form, when compared to the commercial version serratiopeptidase and other control proteins viz—
lysozyme (14.4 KDa) and bovine serum albumin (66.4 KDa) shows the protein elutes at 7.5 min coinciding with the significant peak of commercial
version serratiopeptidase. The elution profile of the recombinant version is identical to the commercial counterpart, except there are very less
intensity minor peaks in comparison to the commercial version. It suggests the protein does not have or very fewer contaminants or degraded
products, if any, in comparison to the commercial counterpart. Comparative c circular dichroism spectra (200–250 nm) and d intrinsic protein
fluorescence spectra(300–450 nm) of recombinant version mature serratiopeptidase (solid line) and its commercial counterpart (dotted line)
showing both versions have an almost identical conformational signature, and recombinant version may prove a better biosimilar for application
purposes
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Page 11 of 15
Srivastavaetal. Microb Cell Fact (2019) 18:215
gene product in the form of insoluble inclusion bodies.
In the present work, we specifically tried to explore the
recombinant expression, purification, and physicochemi-
cal comparison of an industrially and therapeutically rel-
evant broad specificity extracellular metalloprotease of
S. marcescens (MTCC7298) known as serratiopeptidase.
A recombinant expression plasmid pMSrp exclusive to
the expression of mature serratiopeptidase lacking 16
amino acid long N-terminal propeptide was constructed
by cloning the mature serratiopeptidase specific gene
under T7 promoter in pET23b(+) vector plasmid. Trans-
formation and expression in different E. coli expression
host systems confirmed the presence of the gene is toxic
for the cells causing either unsuccessful transforma-
tion (BL21(DE3) cells), lack of expression (BL21(DE3)-
pLysS cells) or expression of the degraded product
(Rosetta(DE3)-pLysS cells). Only E. coli C43(DE3)cells,
engineered specifically for the expression of membrane
proteins and toxic proteins, were expressing the protein
correctly in the form of intracellular insoluble deposits,
i.e., inclusion bodies.
Further optimization of various process parameters
resulted in about five times more expression of serratio-
peptidase than unoptimized conditions (by densitometric
analysis). e overexpressed mature version of serratio-
peptidase protein forms 60–65% pure inclusion bodies.
Solubilization, refolding, and purification provides puri-
fied (> 98%) 45–50mg functionally active protein from
one-liter culture. e discussed recombinant approach
could be a better alternative to the present traditional
production strategy, considering the health hazards asso-
ciated with wild strains of S. marcescens. e biological
identity, activity, and biophysical comparison with com-
mercially available native serratiopeptidase suggest the
recombinant version could serve as a potential biosimilar
for pharmaceutical and variety of industrial applications.
Methods
Screening ofserratiopeptidase producing Serratia
marcescens strain
Serratia marcescens strain (collection id-7298), collected
from the Microbial Type Culture Collection (MTCC),
IMTECH Chandigarh was screened for the extracellular
secretion of serratiopeptidase. 0.2 μl overnight grown
seed culture of S. marcescens was point inoculated on
1% skimmed milk agar plate following 48-h incubation
at 37°C. e identity of protease in secretion was con-
firmed by SDS-PAGE and further by In-gel trypsin diges-
tion and Peptide mass fingerprinting using MALDI-TOF
mass spectroscopy.
Gene amplication andrecombinant cloning
Mature serratiopeptidase gene-specific forward and
reverse primers (shown in Table3) were used for PCR
based amplification of the mature serratiopeptidase
(Msrp) gene, which lacks the initial 48 nucleotides encod-
ing N-terminal propeptide. In brief-e reaction mixture
contained 200µm dNTPs (New England Biolabs, USA),
1× high fidelity buffer (ermo Scientific, USA), and 4.5
units of Phusion polymerase (ermo Scientific, USA)
and 0.2ng/μl genomic DNA of S. marcescens mtcc7298
as template DNA. Amplification reaction comprised of
an initial 3min denaturation (95°C), 35 cycles of 30s
denaturation (95°C), 30s annealing (Ta = 59°C), and 90s
extension (72°C) each, followed by a final 10min exten-
sion (72°C). A control reaction was put together contain-
ing all the components except the template DNA.
Amplified PCR product and empty pET23b(+) vector
were digested for 6h by restriction enzymes NdeI and
XhoI (New England Biolabs, USA). Purified PCR product
and vector after digestion were allowed to ligate at 16°C
for the overnight duration by T4 DNA ligase (New Eng-
land Biolabs, USA). e ligated product was transformed
into competent E. coli DH5α and plated on ampicillin
(200µg/ml) agar plates. Positive transformants contain-
ing the ampicillin-resistant gene (Ampr) were verified
through colony PCR.
Restriction digestion of the recombinant plasmid was
carried out to verify the size and successful insertion of
msrp in the expression vector by visualizing the frag-
ment, equivalent to the size of the cloned gene on 1%
agarose gel. Sequencing of the expression plasmid was
performed to verify the sequence using T7 promoter and
terminator specific primers (Base Asia, Singapore).
E. coli strain optimization
Different E. coli BL21(DE3) based expression host cells
were screened to find an optimal expression host for
pMSrp expression. Freshly prepared CaCl2 chemical
competent cells (1.3 × 107) of four different E. coli expres-
sion strains, viz—E. coli BL21(DE3), E. coli C43(DE3),
E.coli BL21(DE3)-pLysS and E. coli Rosetta(DE3)-pLysS
were transformed by 10ng of pET23b(+) null vector and
recombinant plasmid pMsrp. e positive transformants
were enumerated in each strain, and the number of viable
cells was taken as a criterion to select the optimal host
strain. e number of transformants in E. coli DH5-α was
taken as a positive control. Strains found to be success-
fully transformed with pMSrp were screened for opti-
mal protein expression. Transformed cells of different E.
coli expression strains were grown at 37°C 220rpm and
induced by the addition of 1mM IPTG (SRL, India) when
the optical density of the culture at 600nm reached to
0.6–0.8. Bacterial cultures were allowed to grow for 2h at
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Page 12 of 15
Srivastavaetal. Microb Cell Fact (2019) 18:215
similar growth conditions before visualizing overexpres-
sion through SDS-PAGE.
Protein expression andsolubility assessment
Protein overexpression was visualized by loading normal-
ized amount of the induced, and uninduced cell lysates
on a 12% SDS_PAGE and viewed after coomassie stain-
ing. e level of overexpression was quantified through
the densitometric analysis of the Gel bands. To assess
the solubility of the overexpressed protein, collected cell
pellets after 2 h of induction were lysed by sonication
in resuspension buffer (25mM Tris, 100mM NaCl, and
pH 7.6). e insoluble content of the lysate was sepa-
rated from the soluble part by centrifugation at 10,000×g
(10min), 4°C. e separated pellet from supernatant was
dissolved in an equal volume of resuspension buffer sup-
plemented with 6M urea. Normalized amount of unin-
duced, induced cell lysates, soluble and pellet fraction
was loaded on SDS-PAGE and analyzed after coomassie
staining of the gel.
Optimization ofphysicochemical parameters
For maximal expression of mature serratiopeptidase
in the opted E. coli expression strain, different phys-
icochemical parameters, viz—growth media, Point of
induction, Inducer concentration, the temperature dur-
ing overexpression, post-induction duration were opti-
mized. e optimal value for maximal expression was
determined by variating one parameter at a time. Protein
expression quantification at various points of process
parameters was performed through densitometric analy-
sis of separate lanes of SDS-PAGE representing different
values of the physicochemical parameter under study.
Densitometry analysis
Coomassie blue-stained gels were imaged on Bio-Rad
XR+ (USA) gel documentation unit and analyzed by the
Image-lab program (Bio-Rad, USA). e relative intensity
of the band was measured to quantify the overexpression
of the protein. e relative percentage of the expressed
recombinant protein in whole cell lysate at particular
conditions was measured by selecting the whole lane and
detecting the band through the ‘add band’ option (band
detection sensitivity was high: 75%). A relative compari-
son of the overexpressed protein in different conditions
was performed manually. e protein bands represent-
ing mature serratiopeptidase in each lane were selected
through ‘add band’ option (band detection sensitivity was
high: 75%), and the area was trimmed using ‘adjust band’
option to minimize the background. e quantity of the
selected band was measured through ‘quantity tools’ and
selecting one of the overexpressed protein band (lowest
range) as reference. e intensity obtained was preferred
as criteria to determine the optimal condition for the
maximal overexpression of recombinant mature serratio-
peptidase. At least three independent gels for each con-
dition were analyzed densitometrically, and the relative
mean value was plotted.
Growth prole andspecic growth rate
Escherichia coli C43(DE3) cells alone and transformed
with pMsrp were grown at 37 °C, with and without
induction under shaking condition at 220rpm. Aliquots
of 500μl culture were withdrawn at 30min interval until
12h for optical density measurements. e turbidity of
the samples were measured at 600nm using Beckman
UV-Spectrophotometer (USA). 1mM IPTG was added in
samples representing induced condition when the optical
density of cultures at 600nm reached 0.5–0.6. To calcu-
late the specific growth rate constant, µ, the exponential
(or logarithmic) growth phase was preferred, during this
phase, the rate of increase in the number of cells was pro-
portional to the number of bacteria present at that time.
e specific growth rate constant µ was determined by
fitting the data into the exponential equation using systat
sigmaplot 14.0.
Preparation ofinclusion bodies, refolding, andpurication
Escherichia coli Bl21 C43(DE3) cells were harvested
after 6h of induction at 37°C 220rpm by centrifuga-
tion at 10,000×g for 10min at 4°C. Separated cells were
resuspended in resuspension buffer (Tris: 50mM, NaCl-
350 mM, Beta-mercaptoethanol-5 mM pH-8.0) sup-
plemented with 500μg/ml lysozyme and lysed through
sonication (Qsonica, Cole-Parmer USA) at 25% ampli-
tude (10s ON 50s OFF). e supernatant is separated
from the insoluble pellet by centrifuging the solution at
15,000×g for 20min at 4°C. e separated pellet repre-
senting the mature serratiopeptidase inclusion bodies
were washed twice with wash buffer (Tris: 50mM, EDTA:
5mM NaCl-500mM, Glycerol: 2%, Beta-mercaptoetha-
nol-5mM, Triton-X-100:1.5%, Urea: 2.5Molar pH-6.8).
Remaining detergent was removed by further washing
the inclusion bodies with tris buffer (50mM, pH 7.4) and
stored at −80°C until purification.
Table 3 Mature serratiopeptidase specic PCR primers
used forgene amplication
Oligonucleotides Sequence Restriction site
Msrp_fwd_7298 5′-TAT AAT AC T CAT ATG
GCC GCG ACA ACC -3′
NdeI
Msrp_rev_7298 5′-ATG TAC CTC GAG TTA
CAC GAT AAA GTCC-3′
XhoI
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Page 13 of 15
Srivastavaetal. Microb Cell Fact (2019) 18:215
Inclusion bodies dissolved in 1 ml denaturing buffer
(Tris: 50 mM, NaCl: 500 mM, 6 Molar Guanidinium
hydrochloride, pH 7.6) were refolded by dialysis against
ice-cold refolding buffer (Tris: 25 mM, NaCl: 100mM,
CaCl2: 5mM, ZnCl2: 1mM pH 7.6). e refolded pro-
tein fraction was separated from any misfolded/pre-
cipitated protein by centrifugation and filtration using a
0.2µM syringe filter (MDI, India). Remaining impurities
and traces of denaturant were removed through superdex
G-75 Hi-Prep 10/300 GL gel filtration column (GE Life
Sciences) in refolding buffer lacking the CaCl2 and ZnCl2.
e purity of the fractions collected during elution was
assessed through activity assay, SDS-PAGE, and Coomas-
sie staining.
In‑gel trypsin digestion andpeptide mass ngerprinting
(PMF)
e protein band corresponding to the molecular weight
of serratiopeptidase was manually excised, chopped
into small pieces, and submerged in 25 mM NH4CO3
(Sigma Aldrich USA) containing 25 ng/μl MS grade
trypsin (Pierce, ermo Scientific USA). Digested pep-
tides were extracted in a 1:1 mixture of 0.1% Trifluoro-
acetic acid (Sigma Aldrich, USA) and Acetonitrile (Sigma
Aldrich, USA), mixed with matrix solution and spotted
on MALDI target plate. Generated peptide mass spec-
tra were searched in the Mascot software search engine
(Matrix Science, UK) [46].
Yield andactivity measurements
e yield at different stages of refolding and purification
was measured through Bradford assay, as mentioned by
Kruger in a microplate format [47]. e activity of the
commercial serratiopeptidase and recombinant serra-
tiopeptidase was measured by a protease activity assay as
suggested by Ruchel etal. [48] with slight modifications.
Briefly, the 400μl reaction mixture containing 1% azoca-
sein (SRL, India) and suitably diluted protein was incu-
bated at 37°C for 30min. 150μl of 20% TCA (SRL, India)
was added to stop the reaction and centrifuged for 5min
at 10,000×g. e supernatant was added in an equal
volume of 1N NaOH (Millipore Sigma, USA), and the
absorbance was measured at 450nm. An increase of 0.1
absorption unit after 30min of incubation at 37°C was
taken as one enzyme unit (EU).
Native PAGE andHPLC analysis
Native PAGE analysis was done to assess the homoge-
neity and purity of the purified recombinant version
mature serratiopeptidase. 10% non-denaturing gel lanes
were loaded with mature version serratiopeptidase
along with control samples of BSA. e gel was run
at constant voltage (80V) at 4°C and visualized after
coomassie staining.
Analytical HPLC of 20µl samples containing bovine
serum albumin (BSA), lysozyme, recombinantly pre-
pared, and commercial serratiopeptidase each contain-
ing 5µg protein were run was carried out on Bio SEC-5
HPLC column (Agilent Technologies, USA). e elu-
tion profile for each protein was used for further com-
parison and analysis.
Circular dichroism anduorescence emission spectra
e secondary and tertiary structure profile of the puri-
fied and refolded mature version recombinant serratio-
peptidase was analyzed through circular dichroism and
fluorescence spectroscopy. In brief, one micromolar
recombinantly prepared serratiopeptidase was scanned
in Far-UV circular dichroism spectra, i.e. 200–250nm
in 1mm path length cell using J-810 spectropolarime-
ter (Jasco, UK) flushed with nitrogen gas at 25°C. Sam-
ples were scanned at a rate of 50nm/min with a step
size of 1nm. Spectra were averaged over three scans
and corrected for background by subtracting the scans
of the buffer without protein.
ree independent intrinsic tryptophan fluorescence
emission spectra of one micromolar protein were col-
lected between 300 and 450 nm after excitation at
280nm at 25 °C using carry eclipse fluorescence spec-
trophotometer (Agilent technologies USA) and averaged
out. e circular dichroism profile and intrinsic fluores-
cence spectra in the same range given by one micromolar
commercial version serratiopeptidase (Systopic Labora-
tories, India) were taken as a control for comparison.
Supplementary information
Supplementary information accompanies this paper at https ://doi.
org/10.1186/s1293 4-019-1267-x.
Additional le1. Additional table and figures.
Abbreviations
BSA: bovine serum albumin; CD: circular dichroism; E. coli: Escherichia coli; IPTG:
isopropyl β-
d
-1-thiogalactopyranoside; pMSrp: plasmid for mature serratio-
peptidase expression; msrp: mature serratiopeptidase gene; TFA: trifluoroacetic
acid; TCA : trichloroacetic acid; RDP: Rosetta (DE3)-pLysS.
Acknowledgements
VS acknowledges UGC, Govt. of India for awarding fellowship for the doctoral
research SM acknowledges DST for the scholarship received for junior research
fellow position. Authors acknowledge Prof. U. C. Banerjee, National Institute of
Pharmaceutical Education and Research, Mohali Chandigarh, for helping us
out in the procurement of Serratia marcescens mtcc7298 strain. The authors
acknowledge the efforts made by Jon Tally, Kansas City, USA, for carefully read-
ing the manuscript.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 14 of 15
Srivastavaetal. Microb Cell Fact (2019) 18:215
Authors’ contributions
VS and TKC planned the work. VS and TKC conceived and designed the experi-
ments. VS and SM executed all the experiments. VS, SM, and TKC analyzed the
results, compiled, reviewed, and revised the manuscript. All authors read and
approved the final manuscript.
Funding
No financial aid has been received from external funding agencies for execut-
ing the work. The authors acknowledge the infrastructural support from the
Indian Institute of Technology, Delhi, India.
Availability of data and materials
(1) The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request. (2) All data generated
or analyzed during this study are included in this published article.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
An Indian patent application (201811029173) has been filed for the technol-
ogy/invention disclosed in the present work.
Received: 9 August 2019 Accepted: 6 December 2019
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