Purification and characterization of 37 kDa serine
protease zymogen from silkworm, Bombyx mori
Present by
Mr.M.Kannan, M.Sc., PGDEG,
Research Scholar
Under the guidance of
Prof. M.Krishnan
Insect Molecular Biology laboratory
Department of Environmental Biotechnology
Bharathidasan University, Tiruchirappalli-620 024
Tamil Nadu, India
PROF.M.KRISHNAN’S LABORATORY ACHIEVEMENTS ON
MOLECULAR ENTOMOLOGY
Benefits to sericulture industry
Dr.K.M. Subburathinam: A study on the hydrolyzed soy protein supplementation
to the mulberry silkworm, Bombyx mori
Dr.X.Nirmala : Control of silk gene expression by dietary proteins in the fifth
instar larvae of Bombyx mori
Dr. Prasanta Kumar Kar: Molecular ecology and genetics of wild tasar silk worm
(Antheraea mylitta Drury) in Similipal Biosphere Reserve
Dr.Nitin Kumar Singh: Vitellogenin from the Silkworm, Bombyx mori: An
Effective Anti-Bacterial Agent
Dr. V. Arun Prasanna: Molecular characterization of Bmor 56 receptor from
Silkworm Bombyx mori
Benefits to Insect Pest Management programme
Dr. U. Balasubramaniam: Studies on the toxic effects of insecticides,
monocrotophos and cypermethrin on the mulberry silkworm, Bombyx mori
Dr. A. Rajathi : Studies on the effects of a non-steroidal ecdysteroid agonist
methoxyfenozide (RH-2485), on mulberry silkworm Bombyx mori
Dr. P. Muthu Meenakshi: Studies on the molecular characteristics of insect
vitellogenins and vitellogenin receptors in cotton pest, Spodoptera litura
Dr. R.Chandrasekhar: Expression and sequestration of differentiated fat body
tissues of groundnut pest, Amsacta albistriga Walk
Taxonomy
 Silkworm, B.mori is used for the study of physiology, development and
molecular biology of other harmful insect pests, also act as a model to
study the human disease (Krishnan and Konig, 2010).
 Life cycle of Bombyx mori is around 41±2 days.
 It consist four stages like egg, larva, pupa and adult.
Classification
Kingdom : Animalia
Phylum : Arthropoda
Class
: Insecta
Order
: Lepidoptera
Family : Bombycidae
Genus : Bombyx
Species : mori
Krishnan and
Konig, 2010
Major organs in the larvae of B.mori
Gut
Introduction
 The larval midgut is formed of an epithelial cell monolayer composed of
columnar, goblet and stem cells (Cermenati et al., 2007). The columnar
cells are mainly responsible for food digestion and nutrition absorption.
 Midgut is also a barrier for the foreign substances during food digestion.
It has been found that some proteins such as lipase, 28 kDa serine
protease and SP-2 in midgut have antiviral activity against Bombyx mori
nuclear polyhedrosis virus (BmNPV) (Ponnuvel et al., 2003; Nakazawa et
al., 2004).
 Moreover, midgut has been recognized as one of the important targets for
insect
control
using
B.
thuringiensis
and
silencing
the
expression of vital genes using RNA interference (RNAi). These two are
major tool in insect management (Hakim et al., 2010).
Introduction cont…
 The midgut shows dramatic changes during transition from larva to pupa and
adult
 The pupa is the intermediate phase (Anti-feeding stage), when larval specific
tissues such as fat body, silk gland and larval muscle undergo histolysis and
histogenesis for grow into the organs and external structures of the adult
(Wigglesworth, 1967).
 Proteolytic enzymes are key player in the degradation of structural
components in larval tissues, as they are remodelled during metamorphosis
(Law et al., 1977; Natori, 1999).
 To date, several types of proteases have been studied with respect to their
participation in this midgut tissue remodelling based on their localization and
stage-dependent expression. .
Cathepsin-like proteases (Cathepsin L-like enzyme and cathepsin-D
like enzyme )
Matrix-metalloproteases (MMPs), Serine proteases and caspases
Introduction cont…
 Particularly, serine protease is involved in the breakdown of extracellular
matrix in normal physiological processes, such as embryonic development,
blood coagulation, immune responses, signal transduction, hormone activation,
reproduction and tissue remodelling in insects (Nakajima et al., 1997).
 Previous studies shows that 37kDa serine protease was synthesized as a
zymogen at larval stage and activated upon pupation and involved in midgut
tissue remodelling of silkworm B.mori (Kaji et al., 2009).
 However, further role and molecular mechanism of 37kDa serine protease
activation on midgut tissue remodelling in silkworm, B.mori is unclear.
 In the present study, we plan to characterize the 37kDa serine protease
zymogen activation for better understanding insect gut physiology and exploit
the active 37kDa serine protease as a drug to earlier activation of larval midgut
tissue remodelling (lysis), that will pay a novel bio control strategy.
Objectives
Purification and characterization of the 37 kDa serine protease of
silkworm, Bombyx mori
Prediction of domain, cleavage site of signal peptide and propeptide for
37kDa serine protease
Molecular cloning and over expression of p37kDa serine protease
zymogen of silkworm, Bombyx mori in E.coli
Extraction and purification of p37kDa serine protease zymogen
Proteomic characterization of purified 37 kDa serine protease
Molecular cloning and over expression of active serine protease (Ap37k)
for insecticidal activity
Material and methods
 Domain, Signal peptide and pro-peptide cleavage site detection were
performed using SMART, signal P 4.1 tool and Prop 1.0 server respectively
(Schultz et al. 1998; Duckert et al., 2004).
 Molecular cloning and over expression (Sambrook et al., 1989; Novagen 10th
edition of pET system manual).
 Extraction and purification of over expressed 37kDa serine protease by
using immobilized affinity chromatography (Sambrook et al., 1989).
 Dialysis of purified protein (Sambrook et al., 1989).
 Quantification of purified protein by Bradford assay (Bradford, 1976).
 SDS-PAGE analysis of purified 37kDa serine protease (Laemmli, 1974).
 2-D PAGE analysis of 37kDa serine protease (2DE-DIGE manual, GE heath
care).
 MALDI-TOF/MS analysis and database searching (IISc, Bangalore, India).
Results and discussion
37kDa serine protease precursor
MKWPVIMICLVGWSSCYTQRPIGQKDKGFIDWINNLLGGTTTTTTLRPIDDPPEDC
PSCQCGIARTRRRIVGGYETKETEYPWMAALLYGGRFYCGGALISDLYVLTAAHCT
SGFRKERITVRFLEHDRSKVNETKTIDRKVSDIIRHLRYNPGTYDSDIALLKLAER
VDLSSALKRVRSEGDNGTATDDDKDVGLRPVCLPSSGLSYNNYTGVVTGWGTTEEG
GSVSNALQEVKVPIVTNEECRKGYGDRITDNMICAGEPEGGRDACQGDSGGPMHVL
EMETSKYSEVGVVSWGEGCARPNKPGVYTRVNRYLTWIKQNTRDACNCQ
Theoretical pI/Mw: 6.46 / 36437.12
SMART analysis of 37k serine protease of B.mori
MKWPVIMICLVGWSSCYTQRPIGQKDK
GFIDWINNLLGGTTTTTTLRPIDDPPEDC
PSCQCGIARTRRRIVGGYETKETEYPWM
AALLYGGRFYCGGALISDLYVLTAAHCTS
GFRKERITVRFLEHDRSKVNETKTIDRKVS
DIIRHLRYNPGTYDSDIALLKLAERVDLSS
ALKRVRSEGDNGTATDDDKDVGLRPVCL
PSSGLSYNNYTGVVTGWGTTEEGGSVS
NALQEVKVPIVTNEECRKGYGDRITDNM
ICAGEPEGGRDACQGDSGGPMHVLEMET
SKYSEVGVVSWGEGCARPNKPGVYTRV
NRYLTWIKQNTRDACNCQ
Prediction of signal peptide by Signal P- 4.1
The signal P 4.1 server showed predicted signal peptide site at 18th aa
in p37k
Prediction of signal peptide and propeptide
using ProP 1.0
ProP 1.0 predicts that removal of propeptide at 68aa region in the sequences
leads to formation of mature protease.
Signal peptide
Pro-peptide
Mature protein
1-18
19-68
69-329
Bombyx mori 37-kDa protease (P37k), mRNA
NCBI Reference Sequence: NM_001135203.1
GenBank Graphics
>gi|206725502|ref|NM_001135203.1| Bombyx mori 37-kDa protease (P37k), mRNA
TGCGAGCATCGCGGTGGTCAAAGTCGCTCGCCGTCCTTGATTTCGGGCCTTGAATACGTACGCGTTGGTGTTATA
GATCTCTGCATATCGTCAATCGATATTTTGTTATCAACAATGAAATGGCCAGTGATTATGATCTGCCTGGTTGGTTG
GTCGAGCTGCTACACCCAGCGGCCCATCGGTCAGAAGGATAAAGGATTTATAGACTGGATCAACAATCTCCTTGG
CGGCACAACGACTACCACGACTTTAAGACCTATAGACGACCCGCCCGAGGACTGCCCAAGCTGTCAATGCGGC
ATAGCACGCACTCGTCGGCGCATCGTGGGCGGATATGAAACGAAAGAGACGGAGTACCCCTGGATGGCCGCTCT
TTTGTACGGCGGAAGATTCTATTGTGGTGGTGCACTTATCAGTGATCTGTACGTTTTGACAGCTGCTCATTGTACT
TCAGGATTCCGCAAGGAACGGATTACAGTTCGGTTCTTGGAGCACGATCGTTCTAAAGTAAACGAAACTAAAAC
GATAGACAGAAAGGTGTCTGACATCATTCGTCATCTGCGGTATAATCCCGGAACTTACGACAGTGATATCGCCCTT
TTAAAACTAGCTGAGAGGGTAGACCTCAGCAGTGCATTGAAGCGAGTTCGCAGTGAAGGAGACAATGGCACTG
CCACGGATGACGACAAGGACGTCGGGCTAAGACCGGTCTGTTTACCCAGTTCTGGACTCTCCTATAACAATTAC
ACGGGTGTTGTCACAGGCTGGGGAACTACAGAGGAAGGTGGCTCTGTATCCAATGCATTACAGGAGGTGAAAG
TACCGATTGTGACAAATGAAGAATGTCGTAAAGGCTACGGTGATCGGATAACAGATAATATGATTTGCGCTGGGG
AGCCAGAGGGCGGCCGTGACGCTTGTCAGGGAGACTCGGGTGGACCGATGCATGTTCTTGAAATGGAGACATC
AAAATACTCTGAAGTCGGTGTCGTGTCTTGGGGCGAAGGGTGCGCGCGACCAAACAAACCAGGCGTTTATACCC
GTGTCAATCGATACCTCACTTGGATTAAACAGAACACTCGCGATGCCTGCAATTGTCAATAAAAACACTGCTATG
GTTTAAACATCGCACCCTGGTTCCAGATTTTCGTAGGAGGCTTTTAACTAATTGTTCAAATGACAAATTGTACACC
TTTGTTTTACTTCTTGGTGCCTCGTCGTTATCGGAGGTTGACTGATATGTTCTACAAATTGTTTCTCAGATAAATTT
AATTAATGTTTAATATATTATACGATGTATGAAATTTTAATTTATTAAG
Primer used for amplification of 37 kDa protease gene
p37k F: 5’-CCGGAATTCACAATGAAATGGCCAGTG-3’ Eco R1
p37k R: 5’-CCCAAGCTTTTATTGACAATTGCAGGC-3’ Hind III
PCR amplification and Cloning of 37 kDa serine
protease gene into pET30a vector
Kb M
1
10
3
1
2
Kb M
pET30a
1
10
2
pET 30 a
3
p37k gene
1
A
A : Before Digestion
M for DNA ladder
Lane 1 for pET30a Vector undigested
Lane2 for p37k insert undigested
p37k serine protease at
987 bp after digestion
B
B: After Digestion with Restriction enzyme
ECO-R1 and HIND III
M: DNA ladder
Lane 1: pET30a Vector digested
Lane2: p37k insert digested
pET30a Vector Map
Transformed colonies of p37k in the Kanamycin
supplemented LB agar plate
Grown of Kanamycin
resistant positive colonies
in white colour
Confirmation of p37k clone by PCR (A) and Restriction digestion (B)
Kb M
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 N JM101
Kb
10
3
10
3
M
L1
L2
pET30a@ 5422bp
p37k @987bp
p37k 1
at 1300 bp
1
P37k gene was amplified using T7 Promoter and
Terminator Primer
M: DNA size marker (1 kb, Biotool, Spain)
L1: L1, C3 to C10: p37 k clone
M :DNA size marker (1 kb, Biotool,
Spain
L1: uncut pET30b
L2:The pET30b digested by EcoR1 &
Hind III
 After confirmation, the construct was transformed in to E.coli BL21 (DE3) for
over expression
Mechanism of protein expression in
PET series
SDS-PAGE profile for expression of p37k with His-tag in
TCP, SF& ISF
Total Cell protein (TCP)
kDa M
1 2 3 4 5 6
Soluble fraction (SF)
M 1 2 3 4
5 6
Insoluble body fraction (ISF)
M 1 2 3 4
5 6
100
50
Expression
of p37K
20
The induced and un-induced protein extracts in each TCP, SF and ISF were
separated on 12%SDS-PAGE and proteins were stained with CBBR-250
M :Stands for Molecular weight marker
(Purchased from Thermo scientific)
Lane 1: pET Uninduced
Lane2:pET Induced
Lane 3: p37k Uninduced
Lane4: p37k Induced (0.5mM)
Lane 5: p37k Induced (1 mM)
Lane 6: p37k Induced (1.5 mM)
SDS-PAGE analysis of p37k supernatant after Ammonium sulphate
precipitation
kDa
250
M
C
L1
L2
L3
130
95
72
55
35
28
10
M : Protein marker
C : Whole cell protein
L1: extracellular protein precipitated with 30 % NH2SO4
L2: extracellular protein precipitated with 50 % NH2SO4
L3: extracellular protein precipitated with 70 % NH2SO4
Absence of p37k
expression as
extracellular protein
Extraction and Purification of recombinant
37 kDa serine protease
Purified 37kDa serine protease
The purification of 37 kDa serine protease done by using immobilized metal affinity chromatography
(Ni-NTA agarose). The purified products were separated on 12% SDS-PAGE and protein bands were
stained with CBBR-250. L1 1: standard molecular marker; L2: Crude extract; L3: Unbound; L 4-5: 5
wash and 10 wash. The purified p37kDa serine protease was visualized as single band in lane 6 and 7
with molecular weight of 45kDa (37kDa of serine protease+8kDa of N-terminal vector regions).
2D-PAGE analysis of purified 37 kDa serine
protease
Isoelectric focusing
10 M
12 % SDS-PAGE
3
Arrow indicates the obtained spots for purified 37 kDa serine protease at MW 45
kDa with pI approximately at 6.5-6.8. M- Protein marker
x104
1.50
(b)
1655.786
(a)
1756.883
1.25
1.00
2679.321
1788.870
0.75
4499.315
3957.984
3139.434
3217.564
2877.497
3047.459
2711.311
2807.375
2634.356
2411.188
2306.137
2130.988
2038.033
1866.881
2256.129
1719.559
1619.760
1569.817
1684.821
1344.705
1031.602
823.522
0.25
1216.631
877.083
0.50
725.511
Intens. [a.u.]
MALDI-TOF-MS analysis of 2D-protein spot
0.00
1000
1500
2000
2500
3000
3500
4000
4500
m/z
(c)
(a) The MALDI-TOF chromatogram shows a
large broad protein peak at 1655.786.
(b) The protein score 99 matched to 37kDa serine
protease precursor (Bombyx mori).
(c) The figure shows the 42% protein sequence
coverage.
Mature serine protease gene sequence (from p37k) for
amplification
CGCATCGTGGGCGGATATGAAACGAAAGAGACGGAGTACCCCTGGATGGCCGCT
CTTTTGTACGGCGGAAGATTCTATTGTGGTGGTGCACTTATCAGTGATCTGTAC
GTTTTGACAGCTGCTCATTGTACTTCAGGATTCCGCAAGGAACGGATTACAGTT
CGGTTCTTGGAGCACGATCGTTCTAAAGTAAACGAAACTAAAACGATAGACAGA
AAGGTGTCTGACATCATTCGTCATCTGCGGTATAATCCCGGAACTTACGACAGT
GATATCGCCCTTTTAAAACTAGCTGAGAGGGTAGACCTCAGCAGTGCATTGAAG
CGAGTTCGCAGTGAAGGAGACAATGGCACTGCCACGGATGACGACAAGGACGTC
GGGCTAAGACCGGTCTGTTTACCCAGTTCTGGACTCTCCTATAACAATTACACG
GGTGTTGTCACAGGCTGGGGAACTACAGAGGAAGGTGGCTCTGTATCCAATGCA
TTACAGGAGGTGAAAGTACCGATTGTGACAAATGAAGAATGTCGTAAAGGCTAC
GGTGATCGGATAACAGATAATATGATTTGCGCTGGGGAGCCAGAGGGCGGCCGT
GACGCTTGTCAGGGAGACTCGGGTGGACCGATGCATGTTCTTGAAATGGAGACA
TCAAAATACTCTGAAGTCGGTGTCGTGTCTTGGGGCGAAGGGTGCGCGCGACCA
AACAAACCAGGCGTTTATACCCGTGTCAATCGATACCTCACTTGGATT
BmAp37k F: ATTCCATATGCGCATCGTGGGCGGATATGA Nde1
BmAp37k R: CCGCTCGAGAATCCAAGTGAGGTATCGATTG Xho1
Ap37k PCR amplification and cloning
Kb M L1 L2
Kb M L1 L2 L3 L4 L5
10
3
10
3
1
1
pET30b with 5,4 22 bp
Ap37k with 750bp
Ap37k with
750bp
M : 1 kb marker
L1-L5 :gradient temperature
L6: Negative control (Water as
template)
After Digestion with Restriction enzyme
Nde 1 and Xho 1
M for DNA ladder
L1: pET30b Vector digested
L2:p37k insert digested
Ap37k construct in E.coli BL21 (DE3) in LB agar plate
Grown of Kanamycin
resistant positive colonies
in white colour
Confirmation of Ap37k clone by PCR
Kb
M
L1 L2 L3 L4
L5 L6 L7 L8 L9 L10
10
3
1
P37k gene was amplified using T7 Promoter and Terminator Primer
M: DNA size marker (1 kb, Biotool, Spain)
L1: L1-L3, L5, L10-Ap37k clone, other are negative results
Confirmation of Ap37k clone for over expression
Kb M L1 L2 L3 L4
10
3
1
L5 L6 L7
L8
pET30b with 5,4 22 bp
Ap37k Insert released at
750bp
Amplification of
Ap37k with 750bp
Trypsin like serine protease gene was
amplified using gene specific primer
M: DNA size marker (1 kb, Biotool,
L4: pET30b Undigested
Spain)
L5: pET30b Double digested,
L1 and L2: Negative control and Positive L6: Ap37k construct Undigested,
control (p37k)
L7: Ap37k construct single digested with
L3 : clone (Ap37k)
Xho 1
L8: Ap37k construct double digested
with Nde1 and Xho 1
Protease assay
A
B
C
A: pET30b induced; B:Ap37k clone un-induced; C:Ap37k clone
induced
Red arrow indicate the protease activity of Ap37k lysate
Further large scale over expression and purification of active serine
protease (Ap37k) is ongoing in our laboratory for the application of
insecticidal activity
Summary and Conclusion
 Molecular characterization of 37kDa serine protease will be helpful for
the understanding of midgut physiology in silkworm, B.mori
 The present study successfully cloned and over expressed the 37kDa
serine protease zymogen in BL21 (DE3) at optimized temperature (22°C)
and IPTG (1mM) induction.
 The expression of desired protein was observed in soluble fraction
around >40% at 42.5 kDa.
 Over expressed 37kDa serine protease zymogen was successfully
purified by immobilized affinity chromatography using Ni-NTA
agarose resin.
 The purified 37kDa serine protease zymogen molecular weight and pI
was observed as 42.5kDa and 6.4 respectively. This result similar to
theoretical pI/Mw is 6.46/37 kDa.
Summary cont….
 The MALDI-TOF-MS results confirmed that the over expressed
purified protein spot was 37kDa serine protease.
 Mature form of serine protease (Ap37k) was amplified and cloned for
over expression.
 Isoelectric point of ap37k was observed at 5.91. Acidic pH may play a
major role in the p37k activation.
 Further work is ongoing in our laboratory for the identification
zymogen activation factor and access the Ap37k insecticidal activity
 In future, the development of this enzyme as a biopesticide will be
much helpful for insect pest management through a environmental
friendly approach.
Publications
Singh, N.K., B.C.Pakkianathan, M. Kumar, T.Prasad, M. Kannan, S. Kong and
M.Krishnan. 2013. Vitellogenin from the Silkworm, Bombyx mori: An Effective AntiBacterial Agent. PLoS One. 8:1-8 (IF-3.568).
Mani Kannan, Thangaiyan Suganya, Vimalanathan Arun Prasanna, Neelamegam
Rameshkumar and Muthukalingan Krishnan .2015. An Efficient Method for Extraction
of Genomic DNA from Insect Gut Bacteria - Culture dependent. Current Research in
Microbiology and Biotechnology 3(1): 550-556.
M. Kannan, S. Anbalagan, M. Krishnan, K. Muthukrishnan and V. Gokula. 2015.
New Record of the genus Euclea (Lepidoptera: Limacoididae) from South India
revealed by DNA Barcoding. International Journal of Pure and Applied Zoology. 3,
(1): 92-97 (IF-0.2).
Anbalagan, S., Balachandran, C., Arunprasanna, V., Kannan, M., Dinakaran, S. and
Krishnan, M. 2015. A new species of Simulium (Gomphostilbia) (Diptera: Simuliidae)
from South India. Zootaxa 3974, 555–563 (IF-1.1).
Anbalagan, S., Arunprasanna, V., Kannan, M., Dinakaran, S. and Krishnan, M.
(2015) Simulium (Gomphostilbia) (Diptera: Simuliidae) from Southern Western Ghats,
India: two new species and DNA barcoding. Acta Tropica. 149, 94–105 (IF-2.75).
Our team
Prof.M.Krishnan, Mentor
V.Arun Prasanna
N.Pauline
T.Suganya
T. Ramya
P.Malini
N.Jothi
V. Surya aathmanathan
Acknowledgement
I gratefully acknowledge Board of Research in Nuclear Science (BRNS),
Mumbai (Reference No.2010/34/6/BRNS 769/dated 14.06.2010) for their
financial assistance for this Project and JRF.
 I thank our Bharathidasan University, Trichy, TN, DST-FIST, UGCNON-SAP, New Delhi for providing the sophisticated instrumentation and
research facility.
I extend my sincere thanks to CTEP-DBT International Travel Grant award
to participate in the Asia Pacific Biotech Congress 2015 @ Beijing, China.
I thank to Professor Tsunaki Asano, Department of Biological sciences,
Metropolitan University, Japan for providing me the 37 kDa serine protease
EST clone.
I thank to Professor. Heng Wang, Department of Microbiology and
Parasitology, Institute of Basic Medical Sciences, Beijing, China and Dr.
Shailendra K Verma, Division of Microbiology, DRDE, Jhansi Road, Gwalior,
India for their timely help.
I have gained an immense knowledge on
Biotechnological applications and had an
wonderful experience in this conference. Now
the conference gave a wonderful opportunity to
search “Post Doc” position abroad on
Molecular Biology……..
Thanks to organizing committee of this
conference…….
Our favorite insect
Thank You