PURIFICATION OF CAP PROTEINS OF A.

HYPOGAEA AND THEIR EFFECT ON

BRADYRHIZOBIUM DURING ROOT NODULE
SYMBIOSIS

ROOT NODULE SYMBIOSIS LAB,

NATIONAL INSTITUTE OF PLANT GENOME RESEARCH

Submitted by: Project Supervisor:

Pankaj Dhakad, Dr Senjuti Sinharoy
BS-MS, Department of Biological Sciences Staff Scientist III,
IISER Bhopal NIPGR Delhi
DECLARATION OF AUTHORSHIP

I, Pankaj Dhakad, declare that this “Summer Research Internship Report”

and the work conferred in it are my very own.
I confirm that:
• This work was done wholly or mainly while in candidature for a research
internship at NIPGR, Delhi.
• Where I have consulted the published work of others, this is always
clearly attributed.
• Where I have quoted from the work of others, the source is always
given. With the exception of such quotations, this report is entirely my
own work.
• I have acknowledged all the main sources of help.
• Where the report is based on work done by myself jointly with others,
I have made clear exactly what was done by others and what I have
contributed myself.

Pankaj Dhakad

Place:……………………..

Date:……………………...

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PREFACE

This report is a document of all the activities and experiments performed in

Root Nodule Symbiosis Laboratory, National Institute of Plant Genome
Research, Delhi under the guidance of Dr Senjuti sinharoy.
During my summer internship, I have worked on the project work entitled
“Purification of CAP (cysteine-rich secretory protein, Antigen 5,
Pathogenesis-related 1 protein) superfamily proteins of Arachis hypogaea
and their effect on Bradyrhizobium during root nodule symbiosis”.
No established research has yet been conducted on role of CAP proteins in
root nodule symbiosis of A. hypogaea. Therefore, this project was done to
purify CAP superfamily proteins of A. hypogaea and to determine their role in
root nodule symbiosis.

Pankaj Dhakad

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ACKNOWLEDGEMENT

I am very grateful to H.O.D. of the Department of Biological Sciences, IISER

Bhopal for permitting and supporting me to perform this internship.
I would like to thank Dr Senjuti Sinharoy for allowing me to conduct the
experiments in her laboratory and to provide me with a healthy atmosphere
for carrying out the research activities. I would also like to thank the
wonderful people I met at NIPGR and their valuable inputs received from
them starting with Bikash Sir, Drishti mam, Dr Amit sir, Oindrila mam,
Akanksha mam, Deevita mam. Over the years, this knowledge will be a long-
loved and respected memory and I look forward to working in such a fantast
ic institution again.

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CONTENTS

Declaration of Authorship .................................................................................. 1

Preface .................................................................................................................. 2
Acknowledgement .............................................................................................. 3
Purification of CAP superfamily protein and its effects on Bradyhizobium... 5
1.1 Introduction .................................................................................................................................................. 5
1.2 Workflow ...................................................................................................................................................... 8
1.3 Materials and methods ................................................................................................................................ 9
1.3.1 Cloning .................................................................................................................................................... 9
1.3.2 Expression ............................................................................................................................................. 13
1.3.3 Purification ........................................................................................................................................... 16
1.3.4 Effect on Bradyrhizobium .................................................................................................................... 18
1.4 Obervations ................................................................................................................................................ 19
1.5 Results and Conclusion .............................................................................................................................. 22
1.6 Future Expansion ....................................................................................................................................... 22

Bibliography ....................................................................................................... 23

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 PURIFICATION OF CAP SUPERFAMILY
PROTEIN AND ITS EFFECTS ON
BRADYHIZOBIUM

1.1 INTRODUCTION

Groundnut belongs to the Leguminosae family, the Papilionoideae subfamily

and the Aeschynomeneae tribe. Taxonomically, it groups together with
Aeschynomene, Stylosanthes, and Discolobium. There are 22 species in the
genus Arachis, 9 of which are reported to be nodulated. Groundnut has a high
ability to fix symbiotic nitrogen. Compared to other tropical legumes, the
amount of nitrogen stored by groundnut is high.

There are generally two primary kinds of nodules: determinate and

indeterminate. Determinate nodules are spherical and develop from a non-
persistent meristem whereas indeterminate nodules are elongated and
develop from a persistent meristem.

Essentially, two modes of infection were defined: one by root hair

entry/infection thread spreading, and the other by crack entry/intercellular
spreading. The root hair entry mode of infection is best studied and occurs in
most of the legumes (Medicago, Trifolium, Lotus), while crack entry mode of
infection is comparatively less studied and occurs in few legumes
(Arachis, Sesbania, Aeschynomene).

Crack entry infection requires penetration of Bradyrhizobium through

epidermis of root at the axil of lateral and adventitious roots. After that
nodule organogenesis takes place and a functional nodule form.

Nodule organogenesis is depicted in a following figure:

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 A B

Figure: Nodule organogenesis. (A) Different stages of crack entry infection; (B)
Schematic representation of root nodule symbiosis in A. hypogaea

Since, Crack entry mode of infection is comparatively less known; and the
peptides involved in root nodule development are also yet to be discovered.
The discovery of these peptides may answer “how different salient features
of these types of nodules that make this symbiosis look quite distinctive
occurs?”

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 Objective:
Nodule transcriptome analysis during root nodule symbiosis has shown
induction of CAP superfamily genes of A. hypogaea. Also, Promoter-GUS
activity for this superfamily proteins has been observed in root nodule of
A. hypogaea. Now, to further validate these findings determination of their
role in root nodule symbiosis is the next task.

To determine the roles CAP superfamily proteins, Purification of these

proteins was proceeded and Bradyrhizobium sp. SEMIA was treated with
purified protein to check the effect of these proteins.

The desired proteins gene can be cloned in expression vector at multiple

cloning sites with in continuation of 6xHis tag at N-terminal or C-terminal
end.

Ni-NTA affinity chromatography:

The Ni-NTA affinity purification system is intended to purify 6xHis-tagged
recombinant proteins expressed in cells. Nitrilotriacetic acid (NTA), is a
chelating compound which binds to ligand binding sites of nickel ion (Ni+2).
Proteins containing one or more 6xHis tag can bind to Ni-NTA beads present
in matrix with far more affinity than Antigen-Antibody and Enzyme-Substrate
complex. This binding affinity of 6xHis tag to Ni-NTA beads can be used to
purify recombinant protein containing one or more 6xHis tag.

Figure: Binding His

tag with Ni-NTA
beads

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1.2 WORKFLOW

•Choosing a appropriate vector (pET32a).

•Preparation of vector-insert construct.
Cloning •Reading frame was verified by sequencing.

•Transformation into E.coli expression host (BL21 rosetta).

•Induction and expression of target protein.
Expression •Analyzing expression and solubility of protein.

•Batch purification under native conditions.

•Dialysis for protein purification.
Purification •Bradford assay for protein quantification.

•Treatment of Bradyrhizobium with purified protein.

Effect on •Confocal and SEM (Scanning electron microscope).
Rhizobia

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1.3 MATERIALS AND METHODS

The following materials and methods are used in cloning, expression and
purification of proteins:

1.3.1 CLONING

• Primary considerations
A pET vector for expression was chosen, primarily by considering the
following factors:
i. Cloning strategy
ii. Proteins solubility after expression

By considering these factors, we have chosen a translation vector pET-

32a (+), which denotes reading frame relative to the BamH I cloning site
recognition sequence, GGATCC.

Figure: The map for

pET-32a (+).

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 Figure: pET-32a (+) cloning/expression region.

• Polymerase chain reaction (PCR)

To amplify the gene of interest from cDNA of A. Hypogaea.

Procedure: The following protocol was followed to amplify the gene

of interest from cDNA of A. Hypogaea.

Table 1: PCR reaction for a total volume of 20 µL

S.No. Materials Amount

1. PCR buffer 2 µL

2. dNTP 2 µL

3. MgSO4 1.2 µL

4. Forward and Reverse primers 0.8 µL

5. cDNA 0.5 µL

6. KOD enzyme 0.4 µL

7. Water 12.3 µL

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*Samples were loaded in PCR machine and PCR conditions are adjusted
according to primers used. Then, Samples were subjected to gel
electrophoresis using agarose gel to verify that our gene of interest has been
amplified.

• Gel extraction

To purify the PCR product (gene of interest) from agarose gel. PCR
product was purified using the QIAquick Gel Extraction Kit.

Procedure:
i. DNA fragment was excised from agarose gel with a clean scalpel.
ii. The gel slice was weighed in a clean tube. Then, 3 volumes of QG
buffer was added to 1 volume of gel (100 mg ~ 100 µL).
iii. Incubated at 50oC for 10 min until gel slice has been dissolved.
Vortex was done every 2-3 min of incubation to help in dissolving
gel slice completely. Colour of completely dissolved solution was
yellow (similar to QG buffer).
iv. 1 gel volume of isopropanol was added and sample was mixed.
v. QIAquick spin column was placed in a 2 ml collection tube.
vi. To bind DNA, Sample was applied to QIAquick column and
centrifuged at 13000 xg for 1 min.
vii. Flow through was discarded and QIAquick column was placed
back in the same collection tube.
viii. 0.5 ml of QG buffer was added to QIAquick column and
centrifuged for 1 min.
ix. To wash, 0.75 ml of PE buffer was added to QIAquick column and
Centrifuged for 1 min. Flow through was discarded.
x. QIAquick column was again centrifuged for additional 1 min at
13000 rpm. Again, flow through was discarded.
xi. To remove residual ethanol from column, Incubation at 50oC for
8-10 min.
xii. To elute, 50 µl of nuclease-free water was added to the centre of
the QIAquick column and centrifuged for 1 min.
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 • Ligation

To prepare vector-insert construct.

Procedure: The following reaction was set up

Table 2: Ligation reaction for a total volume of 10 µL

S.No. Materials Amount

1. Reaction buffer 5 µL

2. Vector (pJet, pET32a) 0.5 µL

3. PCR product 1 µL

4. T4 DNA Ligase 1 µL

5. Water (Nuclease-free) 2.5 µL

*The reaction mix was incubated overnight at RT.

Figure: pET32a-7SA9A
construct

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1.3.2 EXPRESSION

Recombinant protein expression was approached by introducing the

plasmid that encodes the desired protein into the host cell, growing the
host cells and inducing expression, and ending with cell lysis and SDS-
PAGE analysis to confirm the protein’s existence.

• Transformation

To introduce plasmid (vector-insert construct) into competent E. coli

cells.

Procedure:
i. Competent cells were taken out from -80oC and thawed in ice
(5-10 min).
ii. Plasmid and competent cells are mixed in a 2ml tube. Tube was
kept in ice for 20-25 min.
iii. Heat shock transformation tube by placing the bottom 1/2 to 2/3
of the tube into a 42°C water bath for 45 secs.
iv. Tubes were kept back immediately in ice for 2-3 min.
v. 0.8 ml of LB was added and tube was kept on shaker at 37oC for
45 min.
vi. Transformation mix was plated in a LB agar plate containing the
appropriate antibiotic.
vii. Plates were incubated overnight at 37oC.

• Colony PCR

Colony PCR was done to determine insert DNA in plasmid constructs.

Designed primers are used specifically to amplify target insert DNA
sequence. Amplified DNA sequence can be used to determine if the
construct contains the DNA fragment of interest. These insert specific

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 primers provide both specificity and size of the DNA fragment of
interest.

Procedure:
Table 3: Colony PCR reaction master mix for a total volume of 20 µL

S.No. Materials Amount

1. PCR buffer 10 µL

2. Forward and Reverse primers 1 µL

3. Water 8 µL

i. Single colony were picked by sterile pipette tip and streaked in a

LB plate containing antibiotic.
ii. Same pipette tip was swirled in colony PCR mix.
iii. Samples were loaded in PCR machine and PCR conditions are
adjusted according to primers used. Then, Samples were
subjected to gel electrophoresis using agarose gel to verify that
our gene of interest has been amplified.
iv. Positive transformant colonies are selected and inoculated 5 ml
LB/amp cultures. Cultures were kept overnight at 37oC.

• Inducing protein expression

Protein expression was induced by adding IPTG to a final concentration

of 0.5 mM.

Procedure:
i. In the morning, 50 ml and 100 ml cultures were inoculated by
diluting the overnight culture 1/50 with M9/amp media.
ii. Cultures were kept on 37oC shaking until an OD of 0.6 to 0.8
reached and then, IPTG was added at the concentration of 0.5
mM. At this step, small volumes of cultures were kept to serve as
a noninduced controls.
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 iii. Both induced and noninduced cultures were kept on shaking at
28oC for 16 hr. Aliquots of the cultures were stored for analysis of
protein expression by SDS-PAGE.
iv. Both cultures were harvested by centrifuging at 13000 rpm for 5
min at 4oC, supernatant discarded, and pellets were kept at -80oC
for further analysis.

• Analysis of protein expression

Total protein expression was analysed by SDS-PAGE.

Procedure:
i. Aliquots of induced and noninduced cultures were harvested,
pellets resuspended in 500 µl running buffer and 50 µl loading
buffer.
ii. Samples were heated at 95oC for 5 min, and centrifuged at 13000
rpm for 3-4 min.
iii. Supernatant was taken by avoiding pellet when pipetting.
iv. Different volumes of supernatant with same concentration were
taken, loading dye added and samples were loaded on SDS-PAGE
gel.

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1.3.3 PURIFICATION

Once the vector and host are carefully chosen, production of

recombinant protein is majorly depends on culture conditions and
induction of expression and in this context, strategies used for
purification of protein are also dependent on these conditions.
Therefore, we have accomplished optimal conditions for small-scale
cultures before purifications on larger scale was attempted.

Procedure:
i. Induced cell pellets were resuspended in lysis buffer (approx.
1/10th volume of original cell culture).
ii. Small amount of lysozyme was added and kept for 1-2 hr in ice.
iii. Samples were homogenized by sonication for 6 x 15 sec, with 45
sec pauses on ice between each burst.
iv. To pellet insoluble debris, cell lysate was centrifuged at 14000-
15000 rpm for 20 min 4oC.
v. Supernatant was transferred in a clean tube without disturbing
the pellet, and 100 µl of supernatant was reserved for SDS-PAGE
analysis.
vi. Equilibrated Ni-NTA beads were added in supernatant for binding
at 4oC for 1-2 hr.
vii. Mixture was transferred in a column and flow through was
collected in a clean falcon tube.
viii. Beads were washed 4 times with 2 ml washing buffer and flow
through collected in W1, W2, W3, and W4 labelled 2 ml tubes.
ix. For elution, Beads were eluted 4-5 times with 1 ml elution buffer
and flow through collected in E1, E2, E3, E4, and E5 labelled 2 ml
tubes.
x. For SDS-PAGE analysis, 25 µl of each solution were taken and
samples were heated at 95oC for 5 min.
xi. Then, loading dye added and samples were loaded on SDS-PAGE
gel.

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Lysis buffer

i. 2-3 mM Imidazole
ii. 50 mM Tris buffer (pH 8)
iii. 100 mM NaCl
iv. 3Mm β-mercaptoethanol
v. 1 mM PMSF

Washing buffer

i. 15 mM Imidazole
ii. 50 mM Tris buffer (pH 8)
iii. 100 mM NaCl

Elution buffer

i. 50 mM Imidazole
ii. 50 mM Tris buffer (pH 8)
iii. 200 mM NaCl

Figure: Purification of 6xHis-tagged protein under native conditions.

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1.3.4 EFFECT ON BRADYRHIZOBIUM

To determine the role of purified CAP protein, we treated Bradyrhizobium

with purified protein and observed in confocal microscope and SEM
(Scanning Electron Micrograph).

Procedure:
i. Bradyrhizobium sp. SEMIA growing in log phase were diluted to OD600
= 0.6.
ii. Diluted bacteria were incubated in phosphate buffer containing 8µM of
purified protein for 3 h at 28oC. For control, Diluted bacteria incubated
in only phosphate buffer.
iii. For SEM imaging, bacterial cells were fixed by 2.5 % glutaraldehyde and
dehydrated by serially diluted ethanol.
iv. Samples were analysed by SEM.
v. For confocal imaging, treated and untreated bacteria were PI
(Propidium Iodide) and SYTO13-stained.
vi. Samples were observed in confocal microscope.

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1.4 OBERVATIONS

1. Restriction digestion

Fig: Restriction digestion of pET32a-

7SA9A with Bam H 1 and Hind III.

2. Colony PCR

Fig: Colony PCR, pET32a with 7SA9A gene insert.

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3. SDS-PAGE (After protein purification)

Fig: SDS-PAGE analysis of purified protein (without β-mercaptoethanol

in lysis buffer)

Fig: SDS-PAGE analysis of purified protein (with β-mercaptoethanol in

lysis buffer)

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4. Confocal microscope images showing treated and untreated
Bradyrhizobium.

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1.5 RESULTS AND CONCLUSION

1.6 FUTURE EXPANSION

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BIBLIOGRAPHY

[1] “A handbook for high-level expression and purification of 6xHis-tagged proteins,” [Online]. Available:
https://www.qiagen.com/es/resources/resourcedetail?id=79ca2f7d-42fe-4d62-8676-
4cfa948c9435&lang%E2%80%99=%E2%80%99en. [Accessed 27 June 2019].

[2] J. B. G. K. G. W. C. L. C. M. B. Kyoung-Hee Choi, “A Tn7-based broad-range bacterial cloning and

expression system,” Nature Publishing Group, 2005.

[3] Clonetech, “TaKaRA Bio Inc,” [Online]. Available: https://www.takarabio.com/. [Accessed 24 June
2019].

[4] “addgene blog,” [Online]. Available: https://blog.addgene.org/.

[5] [Online]. Available: https://international.neb.com/.

[6] “pET system manual,” [Online]. Available:

https://lifeserv.bgu.ac.il/wb/zarivach/media/protocols/Novagen%20pET%20system%20manual.pdf.

[7] D. v. R. Fred C. Boogerd, “Nodulation of groundnut by Bradyrhizobium: a simple infection process by

crack entry,” FEMS Microbiology Reviews, 1997.

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