TITLE: A method for enhancing antimicrobial activity of plant and bacteria derived human beta
defensin-1 (HBD-1) and human beta defensin-2 (HBD-2).
FIELD OF INVENTION:
This invention relates to a method for the production of human beta defensin-1 & 2 (HBD-1 &
HBD-2) in plant and bacterial cells having antimicrobial activity.
This invention also relates to antimicrobial activities of plant and bacteria derived human beta
defensins viz. HBD-1 and HBD-2 individually and in combination.
BACKGROUND OF THE INVENTION:
The progressive reduction of effectiveness of the available antibiotics due to rapid
emergence of antimicrobial resistance emphasizes the necessity for the development of new
classes of drugs for the treatment of infectious diseases (Rice, 2003). Furthermore, search for
highly active antimicrobial substances which possess low cytotoxicity are beneficial for
pharmaceutical applications. Presently, as per WHO recommendation, practices of combinational
approaches employing multidrug become popular to combat against resistant microbes (WHO,
2001). Taken all together, antimicrobial peptides (AMPs), alone or in combination with
conventional antibiotics, are attractive candidates to be used as alternative therapeutic agents for
bacterial infections because of their selectivity, speed of action and inherent immunological
compatibility (Hancock, 2001 and 2005). A novel class of human endogenous antimicrobial
peptides, the important components of innate immunity, called defensins has shown great
versatility in their antimicrobial activity against a diverse range of microorganisms including
bacteria, fungi and viruses. Their mode of action seems largely nonspecific and thus chances of
developing resistance against defensin seem to be minimal.

Human defensins are a critical part of innate immune system that provides the first line
antimicrobial barrier for mucosal surfaces such as the surface of the eye, the environmental
interface of gastrointestinal and genitourinary tracts, tracheobronchial tree, lungs and also the
skin (Lehrer et al., 1993; Ganz et al., 1985). Human defensins are small cationic and cysteine
rich peptides with ẞ-sheet structures that are stabilized by three intramolecular disulphide bonds
between the cysteine residues and having molecular masses between 3 to 5 kDa (Zasloff, 2002;
Ganz, 2003). Based on their distribution, sequence homology and the connectivity of six
conserved cysteine residues, human defensins are classified into two subfamilies, a- and p-
defensins. The disulphide linkages of cysteine residues in a-defensins are Cysl-Cys6, Cys2-
Cys4 and Cys3-Cys5, whereas in p-defensins, the linkages are Cysl-Cys5, Cys2-Cys4 and
Cys3-Cys6 (Ganz, 2003). In humans, a-defensins are found mainly in the secretory granules of
neutrophils and other leukocytes (Zeya et al., 1963; Agerberth et al., 2000). p-defensins are
expressed by most epithelial cells, and their expression is often induced by proinflammatory
stimuli and infection. They are present in the mucosal secretions of respiratory, gastrointestinal,
and urogenital tracts, and in inflamed skin (Harder et al., 1997; Stolzenberg et al., 1997; Bal et
al., 1998). β-defensins are also expressed by human dendritic cells, monocytes, and macrophages
(Ganz, 2003).
The details of their mode of action are unclear; however, it has been suggested that the
cationic characteristic and amphipathic nature of defensin molecules allows them to interact
electrostatically with microbial cell membrane whose composition includes negatively charged
phospholipids in contrast to mammalian cells that are made up of largely neutral zwitterionic
phospholipids (Kagan et al., 1990; Epand and Vogel, 1999). This results in the formation of
pores or disruptions of the bacterial membrane, leading to cytoplasmic leakage and cell death
(Shimoda et al., 1995; Harder et al., 2001; Shai, 2002). They are also effective against enveloped
viruses including herpes simplex virus (HSV) and human immunodeficiency virus (HIV-1) and
the mechanism of virus killing may be driven by high hydrophobicity.

Recently, it has become vibrant that defensins can modulate the innate immune system by
activating macrophages but they do not stimulate Toll-like receptors (TLR), which is an
advantage, as that could lead to sepsis (Hancock and Sahl, 2006). (3-Defensins are also
chemotactic for immature dendritic cells, monocytes, macrophages, memory Th cells and
activated neutrophils (Yang et al., 1995; Chertov et al., 1996; Garcia et al., 2001; Niyonsaba et
al, 2004; Rorhl et al., 2010) but the specific receptor has not been identified yet. Several recent
reports demonstrated that defensins may contribute to control the human immunodeficiency
virus-type 1 (HIV-1) by interfering with its replication in vivo (Nakashima et al., 1993;
Quinones-Mateu et al., 2003; Sun et al., 2005). They also provide a link between the innate and
adaptive immune responses to microbial infection. Some recent studies focused on correlations
between the changes in β-defensin expression pattern and the types and/or stages of tumor
development (Donald et al., 2003; Shiba et al, 2003; Young et al., 2003; Markeeva et al., 2005).
Differential gene expression profiles of HBD-1 also could be useful for differentiating the
subtypes of both renal cell carcinoma and prostate cancer (Donald et al., 2003, Young et al.,
2003). Other indications of defensins are that they appear to have a positive impact on wound
healing (Li etal, 2006).
Due to their broad microbicidal spectrum and versatile additional activities (as discussed
above), β-defensins may become potential candidates for the development of useful peptide
based antibiotics for combating against microbial lessions in human and animals. But the
availability of such high valued peptides is one of the major constraints that determine the
feasibility of their wide-spread usage as antibiotics. Different strategies have been developed to
produce these small antimicrobial peptides (AMPs) using recombinant techniques. Till now, all
efforts to obtain larger quantities of active recombinant human defensins from bacterial and
insect cell lines have been moderately successful (Zhang et al., 1997; Feng et al., 2002). Plant is
emerging as a good expression system for production of eukaryotic proteins due to its several
advantages including high productivity, inexpensive system with low contamination risks etc.,
and includes eukaryotic modifications as well. Recently, transgenic rice plant expressing human
alpha defensin gene HD5 was characterized (Huang et al., 2009; US patent no.
PCT/US06/47606).

Considering the above facts we wanted to investigate in detail the antimicrobial
properties of plant/bacteria derived defensin molecules either individually or in combination
using two important defensin molecules, HBD-1 (Valore et al., 1997) and HBD-2 (Liu et al.,
1998). HBD-1 and HBD-2 molecules shares almost identical structure with alike cysteine-
cysteine disulfide bonds but varied in their sequences both at DNA and protein level. HBD-1 and
HBD-1 were expressed under the control of MUAS35SCP and FUASFSCP promoters,
respectively using a binary vector, pKYLX. We have also expressed these two defensins in E.
coli using pET32a+ vector (Novagen). We studied antibacterial activity of these molecules
individually and in combination. Results obtained clearly indicated that both the plant and
bacteria derived defensins works synergistically when used together in a specific
combination/formulation.
OBJECTS OF THE INVENTION:
An object of this invention is to propose production and purification of two human beta defensin
(HBD-1 and HBD-2) proteins in plant and bacteria.
Another object of this invention is to propose independent methods for expressing these; HBD-1
and HBD-2 in plant and bacterial cell.
Still another object of this invention is to propose independent methods for extracting/ purifying
these; HBD-1 and HBD-2 from plant and bacterial cell.
Further object of this invention is to employ two efficient recombinant plant promoters for better
yield of HBD-1 and HBD-2 recombinant proteins in plant.
Yet, another object of this invention is to propose assaying the antimicrobial activity of both
plant and bacteria derived HBD-1 and HBD-2 individually.

Still further object of this invention is to propose assaying the antimicrobial activity of bacteria
derived HBD-1 against methicillin resistant bacterial strain S. aureus.
Further object of this invention is to propose assaying the antimicrobial activity of both plant and
bacteria derived HBD-1 and HBD-2 in combination.
BRIEF DESCRIPTION OF THE INVENTION:
According to this invention there is provided a method for the production of human beta defensin
1 & 2 (HBD-1 and HBD-2) in plant and bacterial cells having antimicrobial activity comprising
the steps of:
molecular cloning of HBD-1 and HBD-2 in plant expression vector comprising efficient plant
promoter,
molecular cloning of HBD-1 & HBD-2 in bacterial expression vector to express as fusion
protein;
subjecting the clones of transgenic plant & bacteria to the step of characterization;
enriching plant derived recombinant proteins;
purifying the bacteria derived HBD-1 and HBD-2.
Assaying the antimicrobial activity of the enriched/ purified proteins individually and in
combination.
Evaluating the antimicrobial activity of bacteria derived HBD-1 against a methicilin resistant
bacterial strain S. aureus.

BRIEF DESCRIPTIONS OF THE ACCOMPANYING DRAWINGS
Figure 1: Sequence information.
(a) Nucleotide sequence of HBD-1 cDNA.
(b) Amino acid sequence of HBD-1.
(c) Nucleotide sequence of HBD-2 cDNA.
(d) Amino acid sequence of HBD-2.
(e) Nucleotide sequence coding for HBD-1 mature peptide.
(f) Amino acid sequence of HBD-1 mature peptide.
(g) Nucleotide sequence coding for HBD-2 mature peptide.
(h) Amino acid sequence of HBD-2 mature peptide.
Figure 2: Evaluation of transgenic plants expressing HBD-1.
(a) PCR analysis of genomic DNA isolated from different transgenic plants. PCR amplified product
(marked by an arrow) represents the amplified DNA fragment of approximately 850 bp long containing
HBD-1 cDNA in combination with rbcSE9 terminator together. Lanes 1-7 represents PCR product
obtained from seven independent transgenic plants.
(b) Reverse transcription PCR analysis performed using cDNA synthesized from total RNA isolated
from different transgenic plant lines showing expression of transgene, HBD-1. Lanes 2-5 represents
reverse transcription PCR product obtained from different independent transgenic plants.
(c) ELISA analysis performed using proteins isolated from transgenic plant expressing HBD-1
transgene and untransformed control.
uc: Untransformed control, +ve: positive control of respective samples, -ve: PCR negative control,
pKMUAS35SCPHBD-l: Transgenic plant expressing HBD-1 under the control of MUAS35SCP
promoter, M: 100 bp DNA marker.
Figure 3: Evaluation of transgenic plants expressing HBD-2.
(a) PCR analysis of genomic DNA isolated from different transgenic plants. PCR amplified product
(marked by an arrow) represents the amplified DNA fragment of approximately 850 bp long containing
HBD-2 cDNA in combination with rbcSE9 terminator together. Lanes 1-4 represents PCR product
obtained from four independent transgenic plants.

(b) Reverse transcription PCR analysis performed using cDNA synthesized from total RNA isolated
from different transgenic plant lines showing expression of transgene, HBD-2, Lanes 1-3 represents
reverse transcription PCR product obtained from different independent transgenic plants.
(c) ELISA analysis performed using proteins isolated from transgenic plant expressing HBD-2
transgene and untransformed control.
uc: Untransformed control, BC: Buffer control, +ve: positive control of respective samples, -ve: PCR
negative control, pKFUASFSCPHBD-2: Transgenic plant expressing HBD-2 under the control of
FUASFSCP promoter, M: 100 bp DNA marker.
Figure 4: Antibacterial activity assay using plant derived HBD-1.
Antibacterial activity of plant derived HBD-1 was evaluated by colony forming unit (CFU) assay
using plant protein extract in different concentration (40µg and 80µg). BC: Buffer control; UC: Protein
extract from untransformed control; HBD-1: Protein extract from transgenic plants expressing HBD-1
under the control of MUAS35SCP promoter.
Figure 5: Antibacterial activity assay using plant derived HBD-2.
Antibacterial activity of plant derived HBD-2 was evaluated by colony forming unit (CFU) assay
using plant protein extract in different concentration (40µg and 80ug). BC: Buffer control; UC: Protein
extract from untransformed control; HBD-2: Protein extract from transgenic plants expressing HBD-2
under the control of FUASFSCP promoter.
Figure 6: Characterization of HBD-1 in bacteria.
(a) SDS-PAGE analysis of bacterial isolated recombinant HBD-1. L1: Molecular weight marker. L2:
HBDl-trxA fusion protein, L3: HBD1-trxA fusion protein digested with enterokinase, L4: purified
protein from pET32a+ vector transformed bacteria.
(b) ELISA analysis using anti-HBD-1 rabbit antibody as primary antibody shows presence of human
p-defensin-1 (HBDl-trxA fusion protein).
Figure 7: Characterization of HBD-2 in bacteria.
(a) SDS-PAGE analysis of bacterial isolated recombinant HBD-2. L1: purified protein from pET32a+
vector transformed bacteria L2: Molecular weight marker, L3: HBD2-trxA fusion protein, L4: HBD2-
trxA fusion protein digested with enterokinase.

(b) ELISA analysis confirming presence of human β-defensin-2 (HBD2-trxA fusion protein).
(c) Immunoblot analysis of digested fusion protein (HBD2-trxA) showing presence of HBD-2 protein
(L2).
Figure 8: Antibacterial activity assay using bacteria derived HBD-1.
Antibacterial activity of bacteria derived HBD-1 was evaluated by colony forming unit (CFU)
assay using enterokinase digested products of bacteria isolated fusion protein (HBD-1-trxA) in different
concentration (20µg and 40µg). EBC: Enterokinase buffer control.
Figure 9: Antibacterial activity assay using bacteria derived HBD-2.
Antibacterial activity of bacteria derived HBD-2 was evaluated by colony forming unit (CFU)
assay using enterokinase digested products of bacteria isolated fusion protein (HBD-2-trxA) in different
concentration (20µg and 40µg). EBC: Enterokinase buffer control.
Figure 10: Antibacterial activity assay using HBD-1 and HBD-2 in combination.
(a) Antimicrobial activity of plant derived defensins in combination: Antibacterial activities were
evaluated by colony forming unit (CFU) assays using different combinations of plant derived HBD-1 and
HBD-2.
(b) Antimicrobial activity of bacteria derived defensins in combination: Antibacterial activities were
evaluated by colony forming unit (CFU) assays using different combinations of bacteria derived HBD-1
and HBD-2.
Figure 11: Antibacterial activity assay against methicillin resistant bacteria, S. aureus.
Antibacterial activity of bacteria derived HBD-1 (digested with enterokinase) against methicillin
resistant bacteria {Staphylococcus aureus) was evaluated by colony forming unit (CFU) assay. Buffer
control: Enterokinase buffer control.

DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to molecular cloning of HBD-1 (Sequence Information a, b; Figure
1) and HBD-2 (Sequence Information c, d; Figure 1) in plant expression vector containing newly
derived efficient plant promoter MUAS35SCP and FUASFSCP.
Major aspects of the present invention are:
One aspect of the present invention relates to molecular cloning of HBD-1 (Sequence
Information e, f; Figure 1) and HBD-2 (Sequence Information g, h; Figure 1) in bacterial
expression vector and to express as fusion protein.
Another aspect of the present invention relates to production of transgenic tobacco plants
(Nicotiana tabacum samsun NN) expressing HBD-1 and HBD-2.
A further aspect of the present invention is the molecular characterization of transgenic tobacco
plants expressing HBD-1 and HBD-2.
Another aspect of the present invention is molecular confirmation of bacterial clone expressing
HBD-1 and HBD-2.
Another aspect of the present invention is further characterization of both transgenic plants and
bacterial clone by ELISA and/or western blot analysis.
Another aspect of the present invention is enrichment of plant derived recombinant proteins.
Still further aspect of the present invention is the purification of bacteria derived HBD-1 and
HBD-2.

Yet other aspects of the present invention are as follows:
(a) Antimicrobial assay using plant derived HBD-1 and HBD-2 individually.
(b) Antimicrobial assay using bacteria derived HBD-1 and HBD-2 individually.
(c) Antimicrobial assay using plant derived HBD-1 and HBD-2 in combination.
(d) Antimicrobial assay using bacteria derived HBD-1 and HBD-2 in combination.
(e) Antimicrobial assay using bacteria derived HBD-1 against methicillin resistant
Staphylococcus aureus bacteria.
Detailed description
The present invention evaluates the scope of plant derived and bacteria derived defensins; HBD-
1 and HBD-2 as potent antimicrobial agent either independently or in combination. Also this
invention describes detail method for raising transgenic tobacco plants expressing HBD-1 and
HBD-2 genes under control of MUAS35SCP and FUAS35SCP promoters respectively. Apart
from this the salient feature of this project are as follows:
• Molecular cloning of HBD-1 and HBD-2 in binary expression vector pKYLX
coupled to MUAS35SCP and FUAS35SCP promoters respectively
• The Agrobacterium mediated tobacco plant transformation for transgenic plant
production, maintenance of transgenic plants in green house till setting of seeds.
• Germination of T0 seeds on selection media. Crude proteins were extracted from
T1 transgenic seedling and enrichment for defensin from crude plant proteins.
• Molecular characterization of transgenic plants expressing HBD-1 and HBD-2
using rDNA technologies; PCR, reverse transcription PCR and plant derived
proteins by ELISA.

• Proteins enriched with HBD-1 and HBD-2 were used for assaying the
antimicrobial activity independently and in combination.
• Results obtained clearly showed significant antimicrobial activity of said plant
derived proteins.
• Also we observed synergistic effect of HBD-1 and HBD-2 (plant derived) when
used in combination.
• We observed 90% and 79% killing of bacteria when plant derived HBD-1 and
HBD-2 were used in 2:1 and 1:1 (w/w) ratio respectively.
• Molecular cloning of HBD-1 and HBD-2 in pET32a+ vector to generate the
clones pET32aHBD-l and pET32aHBD-2 respectively.
• Positive clones carrying pET32aHBD-l and pET32aHBD-2 were screened by
colony PCR and plasmid isolation followed by restriction digestion.
• Transformation of pET32aHBD-l and pET32aHBD-2 into E. coli (BL21) DE3
cells for expression of defensin peptides in bacterial system.
• Induction of bacterial culture with IPTG at 37°C for 6hrs and purification of -
fusion proteins; HBDl-trxA and HBD2-trxA using Ni-NTA column.
• SDS-PAGE, ELISA and western blot analysis of bacteria derived proteins; HBD-
1 and HBD-2.
• Enterokinase digestion of fusion proteins.
• Digested fusion proteins HBDl-trxA and HBD2-trxA were used for assaying the
antimicrobial activity using E. coli (TBI) as the test strain.
• Digested HBDl-trxA fusion protein was also used to assay the antimicrobial
activity against a methicillin resistant bacterial strain, Staphylococcus aureus.
• Results obtained clearly showed significant antimicrobial activity of said bacteria
derived HBD-1 and HBD-2 when used independently.
• Also we observed synergistic effect of HBD-1 and HBD-2 (bacteria derived)
when used in combination.

• We observed 90%, 96% and 89% killing of bacteria when bacterial derived HBD-
1 and HBD-2 were used in 3:1, 1:1 and 1:3 (w/w) ratio respectively.
• We observed 60% clearance of methicillin resistant bacteria {Staphylococcus
aureus) when treated with digested HBDl-trxA protein at a concentration of
EXAMPLE 1
Cloning of HBD-1 and HBD-2 into plant expression vector
The baculovirus expression vector based cDNA clones of HBD-1 and HBD-2 were
obtained by request from Prof. T. Ganz, UCLA. The sequence coding for the full length cDNAs
of HBD-1 and HBD-2 were PCR amplified using sequence specific primers (HBD-1 Fp/HBD-
lRp for HBD-1 and HBD-2Fp/HBD-2Rp for HBD-2) and respective cDNA clones as template
so as to generate XhoI at 5' end and Sacl at 3' end. The PCR products were gel purified and
cloned into the pKYLX (Schardl et al., 1987) based plant expression vectors. The HBD-1 and
HBD-2 were cloned into pKMUAS35SCPGUS (Patro et al., 2012) and pKFUASFSCPGUS
(Ranjan et al., 2012) vectors respectively replacing the GUS gene. The resulting clones were
designated as pKMUAS35SCPHBD-l and pKFUASFSCPHBD-2 respectively.
EXAMPLE 2
Development and evaluation of transgenic plants
Generation of transgenic tobacco plants
Transgenic tobacco plants harboring pKMUAS35SCPHBD-l and pKFUASFSCPHBD-2
were raised following standard Agrobacterium mediated plant transformation protocol (Dey and
Maiti 1999). The plants were maintained under greenhouse conditions (photoperiod: 16/8 h at

220 µmole m-2 s-1, temperature: 28°±3°C, humidity: 70-75%) till setting of seeds. The seeds were
germinated on MS plates containing kanamycin (300mg/liter). The seedlings (21days old) were
used for further study.
Evaluation of transgenic plants
Genomic DNA PCR
Genomic DNA was isolated from leaves of untransformed control and transgenic plants
expressing pKMUAS35SCPHBD-l and pKFUASFSCPHBD-2 gene constructs following
published protocol (Allen et al. 2007). PCR on genomic DNA was performed using gene specific
forward primer and vector specific reverse primer (HBD-1 Fp and HBD-2Fp as forward primers
for HBD-1 and HBD-2 respectively and rbcSE9Rp as the reverse primer; Table 1). PCR products
were analyzed using a 1% agarose gel.
Reverse transcription PCR
Total RNA from untransformed control and transgenic seedlings expressing the gene
constructs pKMUAS35SCPHBD-l and pKFUASFSCPHBD-2 was isolated using Spectrum™
Plant Total RNA Kit (Sigma, Cat # STRN50) and subjected to DNasel (Sigma, Cat # AMPD1)
treatment. An aliquot of 1µg of DNasel treated RNA was used for synthesis of first strand cDNA
using First strand cDNA synthesis kit (Fermantas, Cat # K1612). HBD-1 and HBD-2 specific
PCR were carried out using sequence specific oligonucleotide primers (HBD-1 Fp, HBD-1 Rp,
HBD-2Fp and HBD-2Rp; Table 1).
ELISA
Total protein from untransformed control and transgenic seedlings expressing HBD-1 and
HBD-2 proteins were isolated individually. Briefly, 1gm of seedlings was frozen in liquid
nitrogen and ground to fine powder. The powder was transferred to 1ml of protein extraction
buffer (200mM phosphate buffer, 100mM NaCl, pH 7) containing protease inhibitor cocktail
(Sigma, Cat# P9599) and incubated on ice for 30 mins with intermittent mixing. The samples
were centrifuged at 14000g for 15 mins. Trie supernatant containing the proteins were collected

in fresh tube. Concentrations of proteins were measured according to Bradford (1976). An
indirect ELISA was performed using the total protein isolated according to Vazquez et al. 1996.
Briefly, ELISA plates were coated with 5µg of protein obtained from untransformed control and
transgenic plant. Subsequently, primary antibodies specific to HBD-1 and HBD-2 (Santa Cruz,
CA) were applied for respective transgenic plant samples. Color development reactions were
carried out using horseradish peroxidase conjugated secondary antibody and ortho-
phenylenediamine as substrate. The OD492 was measured.
EXAMPLE 3
Enrichment of plant derived HBD-1 and HBD-2
Approximately 20g of cleaned and frozen leaves were ground to a fine powder with
liquid nitrogen. The powder was homogenized with 60 ml of 50 mM sulfuric acid. After stirring
for 1 h, insoluble material was removed by filtration through miracloth. The resulting filtrate was
centrifuged at 14,000g for 20 min, at 4°C and the supernatant was neutralized to pH 7.8 with 1 M
NaOH. After incubation at 4°C for 30 min, the mixture was centrifuged again at 14,000g, for 20
min, at 4°C. Solid ammonium sulfate was added to the supernatant to obtain 35% relative
saturation. The precipitate, formed after standing for 1 h at 4°C, was removed by centrifugation
at 14,000g, 4°C for 30 min. The supernatant was incubated overnight at 4°C as 80% saturated
ammonium sulfate solution under constant stirring. The precipitate was collected by
centrifugation and dissolved in a small amount of 20 mM Tris-HCl buffer, pH 7.4 and 10 mM
NaCl. The fraction of heat-stable proteins was obtained by heating the re-suspended sediment at
85°C for 10 min and removing of heat-denatured protein precipitates by centrifugation (20,000g,
20 min, 4°C). Supernatant was dialyzed against a buffer, containing 20 mM Tris-HCl pH 7.4,
100 mM NaCl using 2 KDa cutoff dialysis tubing (Sigma). Elution of proteins was monitored by
absorbance at 280 nm. Protein concentration was determined according to Bradford (1976).

EXAMPLE 4
Cloning of HBD-1 and HBD-2 into bacterial expression vector
The sequences coding for the mature peptide of HBD-1 and HBD-2 were PCR amplified
using sequence specific primers (HBD-lAspFp and HBD-1Rp for HBD-1. HBD-2AspFp and
HBD-2Rp for HBD-2; Table 1) and the respective cDNA clones as template so as to generate
Ncol at 5' end and Sacl at 3' end. The PCR products were gel purified and cloned into the
corresponding sites of the bacterial expression vector pET32a+ (Novagen). The resulting clones
were designated as pET32aHBD-l and pET32aHBD-2 respectively.
EXAMPLE 5
Purification of bacteria derived HBD-1 and HBD-2
The pET32aHBD-l and pET32aHBD-2 were transformed into E.coli BL21 DE3 cells
and spread onto LB agar plates containing ampicillin. A single colony from each were inoculated
separately into 10ml of LB broth and grown at 200rpm, 37°C till the OD600 reached to 0.4 - 0.6.
The bacteria were harvested, resuspended in fresh LB and inoculated into 200ml fresh LB in a
1liter flask. The cultures were grown (at 37°C and 200rpm) till the OD600 reached to 0.6 and
induced with 0.5mM IPTG. The bacteria were harvested 6 hrs post induction the by centrifugingl
at 6000g for 15mins. The fusion proteins HBDl-trxA and HBD2-trxA were purified using;
QIAexpress® Ni-NTA Fast Start kit (Qiagen, Cat. no. # 30600) according to supplied protocol.
EXAMPLE 6
SDS-PAGE and immunoblot analysis of purified bacterial protein
The purified proteins; HBDl-trxA and HBD2-trxA were quantified following protocol!
described by Bradford (1976). The fusion proteins were desalted using an Ultracel YM-3

membrane centrifugal device (Millipore, Cat. No. #4202) and subjected to enterokinase digestion
(lU/50ug protein) at 25°C for 16hrs and analyzed on an 18% SDS-PAGE (Laemmli, 1970). For
western blot analysis, the HBD2-trxA fusion protein and corresponding digested protein were
resolved on an 18% SDS-PAGE. The resolved proteins were transferred on to a 0.2(i PVDF
membrane. The membrane was probed with anti-HBD-2 primary antibody followed by
secondary antibody conjugated to horse radish peroxidase. The western blot development
reaction was carried out using ECL detection kit (Amersham).
EXAMPLE 7
Evaluation of antibacterial activities
Using plant derived defensins; HBD-1 andHBD-2 Total proteins isolated from untransformed control and transgenic seedlings (21 days old)
expressing HBD-1 and HBD-2 were used. E. coli (TB1) strain was used as test strain for
antibacterial assays following protocols described by Krishnakumari et al. (2006) with a slight
modification. Briefly, an aliquot of 100 µl from overnight grown bacterial culture (E. coli TB1)
was allowed to grow in 10 ml LB at 37°C till its OD600 reached to 0.5. Subsequently, the bacteria
were harvested and washed with phosphate buffer saline (PBS). The bacteria pellet was
resuspended in 10mM sodium phosphate buffer, pH 7.4. After appropriate dilution (10-4)
resuspended bacteria were incubated with 40µg and 80µg of proteins in a 100µl reaction volume
at 37°C individually with constant shaking at 200rpm. After 3hr of incubation, the samples were
diluted for 50 times and 100µl of each were spread on LB agar plates (in triplicate). After
overnight incubation at 37°C numbers of CFUs were counted.

Using bacteria derived defensins; HBD-1 and HBD-2
Antimicrobial assays were carried out following the protocol as mentioned above using
bacteria derived defensins. Briefly, bacteria were incubated with 20µg and 40µg of HBD1-trxA
and HBD2-trxA fusion protein and enterokinase-digested HBDl-trxA and HBD2-trxA fusion
proteins in 100µl reaction volume at 37°C and 200rpm. 1X enterokinase buffer was used in as
buffer control.
EXAMPLE 8
Antimicrobial activity assay of plant/ bacteria derived HBD-1 and HBD-2 in combination
Antimicrobial activity assay using plant derived defensins; HBD-1 and HBD-2in combination
The antimicrobial assays using different combinations of plant derived HBD-1 and HBD-
2 were carried out similarly as described above using following different combinations:
i) HBDl:HBD2=40µg:20µg
ii) HBD-1: HBD-2=30µg:30µg
iii) HBD-1 :HBD-2=20µg:40µg.
Antimicrobial activity assay using bacteria derived defensins; HBD-1 and HBD-2 in
combination
The antimicrobial assays using different combinations of bacteria derived HBD-1 and
HBD-2 were carried out similarly as described above using following different combinations:
i) HBDl:HBD2=30µg:10µg
ii) HBD-1: HBD-2=20µg:20µg
iii) HBD-1: HBD-2=10µg:30µg.

EXAMPLE 8
Antimicrobial activity of bacteria derived HBD-1 against methicillin resistant strain;
Staphylococcos aureus
An aliquot of 100 µl from overnight grown bacterial culture of Staphylococcos aureus
(resistant to methicillin antibiotic) was allowed to grow in 10 ml LB at 37°C till its OD600
reached to 0.5. Subsequently, the bacteria were harvested and washed with phosphate buffer
saline (PBS). The bacteria pellet was resuspended in 10mM sodium phosphate buffer, pH 7.4.
After appropriate dilution (10-4) resuspended bacteria were incubated with 20µg and 50µg of
HBDl-trxA fusion protein and enterokinase-digested HBDl-trxA fusion protein in 100µl
reaction volume at 37°C with constant shaking at 200 rpm. Enterokinase buffer (1X) was used as
buffer control.

WE CLAIM:
1. A method for the production of human beta defensin 1 & 2 (HBD-1 and HBD-2) in plant and
bacterial cells having antimicrobial activity comprising the steps of:
molecular cloning of HBD-1 and HBD-2 in plant expression vector comprising efficient plant
promoter,
molecular cloning of HBD-1 & HBD-2 in bacterial expression vector to express as fusion
protein;
subjecting the clones of transgenic plant & bacteria to the step of characterization;
enriching plant derived recombinant proteins;
purifying the bacteria derived HBD-1 and HBD-2.
2. The method as claimed in claim 1, wherein said plant promoter used are MUAS35SCP and
FUASFSCP.
3. The method as claimed in claim 1, wherein the said step of characterization of both transgenic
plants and bacterial clone is done by ELISA and/or western blot analysis.
4. The method as claimed in claim 1, wherein said step of molecular cloning of HBD-1 and
HBD-2 in plants expression vector coupled to MUAS35SCP and FUASFSCP promoters
respectively,

extracting crude protein from T1 transgenic seedling and enrichment for defensin from crude
plant proteins;
characterizing transgenic plants expressing HBD-1 & HBD-2 using rDNA technologies PCR
reverse transcription PCR and plant derived protein by ELISA;
assaying the antimicrobial activity independently and in combination.
5. The method as claimed in claim 1, wherein said step of molecular cloning of HBD-1 and
HBD-2 in bacterial expression vector comprising the steps of:
molecular cloning of HBD-1 and HBD-2 in pET32a + vector to generate the clones pET32a
HBD-1 and pET32a HBD-2 respectively,
screening positive clones carrying HBD-1 and HBD-2 by colony PCR and followed by plasmid
isolation and confirmation by NcoV SacI restriction digestion;
transferring pET32a HBD-1 and pET32a HBD-2 into E. coli BL21 (DE3) cells for expression of
defensin peptides in bacterial system;
subjecting the bacterial cultures to the step of induction with IPTG at 37°C for 6 hrs and
purification of fusion proteins HBD1-trxA and HBD2-trxA using Ni-NTA column;
digesting the fusion proteins with enterokinase and assaying the antimicrobial activity using E.
coli as test strain.
6. The method as claimed in claim 1, wherein the combination of plant derived HBD-1 and
HBD-2 at ratio 2:1 and 1:1 that showed significant antimicrobial activity.

7. The method as claimed in claim 1 wherein combination of bacteria derived HBD-1 and HBD-
2 at ratios 3:1, 1:1 and 1:3 that showed significant antimicrobial activity.

ABSTRACT

A method for the production of human beta defensin 1 & 2 (HBD-1 and HBD-2) in plant and
bacterial cells having antimicrobial activity comprising the steps of: molecular cloning of HBD-1
and HBD-2 in plant expression vector comprising efficient plant promoter, molecular cloning of
HBD-1 & HBD-2 in bacterial expression vector to express as fusion protein; subjecting the
clones of transgenic plant & bacteria to the step of characterization; enriching plant derived
recombinant proteins; purifying the bacteria derived HBD-1 and HBD-2; determining the
antibacterial activity of plant and bacteria derived HBD-1 and HBD-2 individually and in
combination; synergistic antimicrobial activities were observed for both plant/ bacteria derived
HBD-1 and HBD-2 when they were used in combination.