DESC:FIELD OF INVENTION
The current invention relates to a method for separating and purifying the serine proteinase/protease inhibitors, Kunitz inhibitor and Bowman-Birk inhibitor from plant parts in an efficient, time-saving, and cost-effective manner. The invention relates to a method for purifying the economically important and multifunctional Kunitz inhibitor and Bowman-Birk inhibitor proteins from seeds of plants, using trichloroacetic acid at an early stage of the purification method.
BACKGROUND
Protease inhibitors (PIs) are widely distributed in living organisms like bacteria, fungi, plants, and humans. The main function of PIs is to regulate the proteolytic activity in organisms. In plants, PIs participate in the regulation of endogenous proteolytic processes. Moreover, PIs also participate in the regulation of cell death during plant development and senescence, mobilization of storage proteins, modulation of apoptosis and programmed cell death, and act as defence proteins or compounds against animals, insects and microorganisms. PIs have been grouped into 48 families and classified into four mechanistic classes i.e. cysteine, serine, metallo- and aspartic inhibitors based on the amino-acid present in the catalytic site of its cognate proteinases . Plant serine PIs are grouped into Kunitz (trypsin) inhibitors (KI or KTI), Bowman-Birk inhibitors (BBI), potato type I, and potato type II inhibitors, cereal trypsin/amylase inhibitors, metallo carboxypeptidase inhibitors, mustard trypsin inhibitor, cysteine protease inhibitors and squash inhibitor families.
Serine proteinase inhibitors or SPIs from plant sources have acquired exceptional importance as natural plant protecting agents. They block digestive proteinases of insects and impart anti nutritional effects. In addition, a number of vital processes including growth and development of insects are also affected by these SPIs. Plant SPIs are capable of inhibiting the extracellular serine proteases produced by phytopathogenic microorganisms, which are necessary to invade plant cells and to supply nutrients. In response to the action of the extracellular proteases released by pathogens, plants induce the expression of SPIs to suppress the growth of microorganisms and control the infection process. Moreover, plant SPIs are capable of suppressing the enzymatic activity in the digestive tract of insects, preventing the assimilation of vegetable proteins.
In modern, highly intensive agriculture, the control of insect pests is mainly achieved with the application of chemical pesticides. Heavy reliance on this sole strategy is associated with several drawbacks, and the development of alternative or complementary methods to chemical control is desirable. Lepidopteran insect pests are the most devastating crop pests in the world. Among them, Achaea janata and Helicoverpa armigera are two important Lepidopteran insect pests. H. armigera is a polyphagous pest feeding on more than 200 varieties of plant species. It attacks not only economically important crops but also many horticultural and ornamental crops. Contrarily, A. janata, the major pest of castor feed on the leaves and defoliate the whole plant leaving only the main stem which leads to death of young plants or reduction in yield of old plants. Controlling these insect pests is a major challenge because they are highly capable in overcoming both host plant’s defence as well as chemical pesticides. Moreover, chemical pesticides are harmful to human health as well environment. Major midgut proteases of these insect pests are reported to be of serine type. Effect of PIs on these insect’s growth and development has been demonstrated by feeding with artificial diet and developing transgenic plants expressing such inhibitors. But, as plant PIs and insect proteases have co-evolved over a long period of time, insects often overcome plant defence by several strategies like (i) over expressing proteases to suppress inhibitory effects of PIs (ii) degrading plant PIs with gut proteases and (iii) synthesizing inhibitor insensitive proteases. In this context, continuous screening of non-host PIs to which insects have less or no prior exposure may become potent tool to identify PIs effective against these pests and to overcome this situation.
There is an urgent need for the development of novel human and environment friendly methods to control these agricultural pests. One promising strategy is the use of plant proteinase inhibitors (PIs).
Kunitz inhibitors (KIs) are proteins of Mr ~20 kDa that contains four cysteine residues forming two disulfide bridges and possess a single reactive site whereas BBIs have Mr ~ 8-10 kDa, with high cysteine content and two reactive sites .Recently, BBIs were reported to show high inhibitory activity towards the serine type gut proteases of Achaea janata, a major castor pest. Further, KIs were reported to show potential inhibition against gut proteases of Helicoverpa armigera, a major pest of cotton, black gram, red gram, tomato. In this scenario, separation of BBI and KI will be more specific and beneficial for integrated pest management. BBIs and KIs are also reported to possess some therapeutic properties such as anti-cancer and anti-coagulant properties.
Protein purification is a very laborious process involving a series of steps such as extraction, precipitation, centrifugation and application of various chromatography methods like ion exchange, affinity and gel filtration, which may take approximately 10 - 15 days to obtain a protein in pure form. Separation of BBI and KI from same seed material is even more difficult process due to the oligomeric nature of BBI and its closer molecular mass with KI, which would take approximately about 10 days.
The current invention relates to a quick extraction method to separate BBIs and KIs from different varieties of seeds, ,from plants such as green gram, red gram and black gram, in 2 to 3 days of time period, instead of 10 days or more that conventional methods take. This quick extraction method is based on the differential solubility of BBIs and KIs in trichloroacetic acid. The BBI and KI proteins purified by the reported procedure exhibit similar biochemical, biophysical, insecticidal properties and have the same activity as that of the PIs purified by other reported methods, while being a highly cost effective, efficient method that can be performed in a much lesser time.
SUMMARY
One Embodiment of the current invention is a method of purifying protease inhibitor proteins Bowman-Birk inhibitor (BBI) and Kunitz inhibitor (KI) from plant seeds, the method comprising the steps of:
(a) making crude extract from plant seeds;
(b) extraction of the crude extract from step (a) with 2.5% Trichloro acetic acid (TCA);
followed by heating and centrifugation to obtain a first pellet fraction and a first supernatant fraction;
(c) performing acetone precipitation with the first supernatant fraction from the step (b) to obtain a second pellet fraction, dissolving the second pellet fraction in a buffer and performing affinity chromatography with the dissolved pellet to obtain BBI protein;
(d) dissolving the first pellet from step (b) in a buffer with pH 8-9 followed by TCA extraction, heating and centrifugation of the dissolved first pellet to obtain a double extracted third pellet fraction; and
(e) dissolving the third pellet fraction in buffer, and performing affinity chromatography of the dissolved third pellet fraction from step (d) followed by adding twice the volume of sodium acetate buffer, heating and centrifugation, subjecting supernatant to acetone precipitation to obtain a fourth pellet containing purified KI protein followed by dissolving the fourth pellet in a buffer to obtain dissolved KI protein.
In one embodiment, the crude extract in step (a) is made by crushing dried plant seeds, into fine powder, followed by depigmentation and defatting with repeated washes of acetone and hexane, respectively.
In one embodiment, the trichloroacetic acid (TCA) used for extraction in step b) is 2 to 3 % TCA.
In one embodiment, the BBI and KI (PIs) are purified from plants of leguminosae, graminaceae and Solanaceae families. In one embodiment, the BBI and KI (PIs) are purified from plants of leguminosae family.
In one embodiment, the BBI and KI PIs are purified from leguminosae family members, examples of which include, but are not limited to, red gram, green gram, black gram, horse gram, soybean, chickpea and peanut.
In one embodiment, the BBI and KI PIs are purified from wild varieties of red gram varieties such as Rhynchosia sublobata and Cajanus platycarpus, and cultivated varieties of red gram.
In one embodiment, the yield of purified BBI using the method disclosed herein is 0.3 to 0.45mg protein per gram dried seeds, with 14 to 26 % yield recovery and 37-55 fold purification, and the yield of purified KI is 0.1 to 0.135 mg per gm seeds with 2.9 to 4% yield recovery and 25-fold purification, varied respectively.
In one embodiment, the purified BBI and KI proteins obtained by the method disclosed herein exhibit oligomerization (self association pattern). In one embodiment, the purified BBI and KI (PIs) have protease inhibitory activity against serine proteases (examples of which include, but are not limited to, trypsin and chymotrypsin).
In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable at pH 2.0 to 12.0. In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable at a temperature up to 100°C. In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable to reducing agents such as Dithiothreitol (DTT).
In one embodiment, the BBI PIs purified by the method disclosed herein exhibit specific activity against trypsin-like gut proteases of lepidopteran insect pest, Achaea janata (A. janata gut trypsin-like proteases ;AjGPs) ((21,000 to 33,000 AjGPI units / mg protein with an IC50 of 22 to 96ng).
In one embodiment, the BBI PIs purified by the method disclosed herein cause significant weight reduction in larvae of A. janata by 76-83% of control larvae weight, mortality rates of larvae by 20-45% and showed inactive larval-pupal intermediates (30-50%) formation during its life cycle as compared with control larvae during their metamorphosis from larva to pupa stage.
In one embodiment, the KI PIs purified by the method disclosed herein exhibit specific activity against trypsin-like gut proteases of lepidopteran insect pest Helicoverpa armigera (18,520 to 26,240 HaGPI units/mg protein with an IC50 of 42 to 150 ng)
In one embodiment, the KI PIs purified by the method disclosed herein lead to significant weight reduction in H. armigera larvae by 66-70% of control larvae weight, mortality rates of larvae by 20-33% and showed inactive larval-pupal intermediates (36-52%) formation during its life cycle as compared with control larvae during their metamorphosis from larva to pupa stage.
In one embodiment, the KI PIs purified by the method disclosed herein (PI) exhibit 33% reduction in the growth of epithelial breast cancer cell lines (MCF-7) as compared with control (MCF-10A) non-cancerous cell lines
In one embodiment, the KI PIs purified by the method disclosed herein lead to 60% inhibition in the growth of methicillin-sensitive Staphylococcus aureus bacteria cells as compared with control cells (without KI/PI).
BRIEF DESCRIPTION OF FIGURES:
Fig.1. Flow chart depicting the step wise separation of BBI and KI from crude extract of mature dry seeds. S, P, AcP and TAC represents Supernatant, Pellet, Acetone precipitation and Trypsin affinity column, respectively.
Fig. 2. Purification pattern of BBI and KI: Elution profile of A) trypsin affinity column loaded with supernatant (S1a + S1b) enriched with BBI B) TAC loaded with Pellet (P3) enriched with KI. Peak I indicates the flow through, peak II with an asterisk (*) indicates the active protein fraction pool. Tricine SDS PAGE gels showing the purification pattern of BBI and KI from green gram (C), Cajanus platycarpus(D) and Rhynchosia sublobata (E), respectively. Lane 1: Protein standard marker, lane 2: crude extract (10 µg), lane 3: S1a (5 µg), lane 4: S1b (5 µg), lane 5: active fraction pool of BBI (2.5 µg), lane 6: P3 (5 µg), lane 7: active fraction pool of P3 (2.5 µg), lane 8: P4 (pure KI-2.5 µg) lane 9: Soybean BBI (2.5 µg). Blue color circles indicates pure BBI and KI fractions.
Table. 1. Purification table showing the purification fold, specific activity and yield recovery of BBI and KI separated from mature dry seeds (10g) of green gram, Cajanus platycarpus (ICPW 68) and R. sublobata.
Fig. 3.In vitro inhibitory activity of BBI and KI against serine proteases trypsin (A) and chymotrypsin(B). Visualization of in-gel activity of BBI (2 µg) and KI (2 µg) against trypsin (C) and chymotrypsin (D) in gelatin SDS PAGE (15%). Soybean BBI (SBBI- 2 µg) was used as reference marker. Bands were visualized after staining with Coomassie Brilliant blue R-250.
Fig. 4. Self association pattern of BBIs and KIs: A) Concentration dependent oligomerization of RsBBI (2, 4 µg) and RsKI (3, 6 µg) (Rs is R. sublobata) on Tricine SDS-PAGE (15%). MALDI-ESI-Q-TOF analysis of RsBBI (B) and RsKI (C)at 5000-60000 m/z. H, M, D, T, Te, P and He indicates halfmer, monomer, dimer, trimer, tetramer, pentamer and hexamer, respectively. MALDI-TOF analysis of Cajanus platycarpus BBI (CpBBI) (D) and GgKI(Green gram Kunitz inhibitor) (E) between 5000-60000 m/z. Inset of D and E showing the zoomed spectra of CpBBI (b/w 5000-8000 m/z) and GgKI (17200 – 19800 m/z) represents the isoinhibitors. Molecular masses of all BBIs and KIs purified by using Mass spectrometry (F).
Fig. 5. Functional stability of BBI and KI against wide range of temperature and pH. The TI and CI activity against temperature was assessed by incubating for 30 min at mentioned temperature (A, B and C). The influence of pH was assessed by incubating at described pH for 30 min(D, E and F) and the effect of DTT was monitored by incubating in different concentrations of DTT for 1 h(G, H and I), respectively. The data represented is mean ± SE of three biological replicates.
Fig. 6. In vitro inhibitory activity of BBI and KI against midgut extracts of Achaea janata and Helicoverpa armigera. Specific activity of BBI and KI against midgut proteases of A. janata(A) H. armigera (B). Visualization of in-gel activity of BBI (2.5 µg) and KI (2.5 µg) against AjGPs (A.janata gut proteases) (C) and HaGPs (D) in gelatin SDS PAGE (15%). Soybean BBI (SBBI- 2.5 µg) was used as reference marker. Bands were visualized after staining with Coomassie Brilliant blue R-250.
Fig. 7. Inhibitory potential of BBI and KI against midgut proteases of A. janata and H. armigera. Half-maximal inhibitory concentration (IC50) of BBIs and KIs against AjGPs (A, B, C) and HaGPs (D, E, F), respectively.
Fig. 8. In vivo inhibitory effect of BBI and KI on growth and development of A. janata: Photographs depicting A) the reduction in size of larvae upon feeding with BBI and KI (8 µg/ cm2) as compared to larvae reared on control leaves. B) reduction in mean body weight of larvae; C) reduction in pupal weights; D) formation of larval-pupal intermediates. E) Table representing the overall growth and development of A.janata upon feeding with BBI and KI from green gram, C. platycarpus and R. sublobata.* indicates the larval-pupal intermediates.
Fig. 9.In vivo inhibitory potential of BBI and KI on growth and development of H. armigera: Photographs depicting A) reduction in size of 5th instar larvae upon feeding with BBI and KI (0.05%) as compared to larvae reared on control diet. B) reduction in mean body weight of larvae; C) reduction in mean pupal weights; D) formation of larval-pupal intermediates. E) Table representing the overall growth and development of H. armigera upon feeding with BBI and KI from green gram, C. platycarpus and R. sublobata. * indicates the larval-pupal intermediates.
Fig.10. Therapeutic properties of KIs: (A) Anti-cancer activity of RsKI against breast cancer (MCF-7 and MDA-MB-231), cervical cancer (HeLa) epithelial cell lines as compared to non-cancerous cell line MCF-10A and (B) Anti-bacterial effect of KIs on the growth of Methicillin-sensitive staphylococcus aureus, a gram positive bacterium. Commercially available Soybean BBI was used as standard in both the experiments.
Detailed Description of Invention
The current invention discloses a novel method for separating and purifying industrially and economically significant Kunitz inhibitor and Bowman-Birk inhibitor proteins from plants. The disclosed method is a rapid, efficient, cost-effective and easy method, compared to the known methods for purifying these proteins. The proteinase inhibitors (PIs) purified by the reported procedure exhibit similar biochemical, biophysical, insecticidal properties and have similar activity levels as that of the PIs purified by other reported methods.
The current invention discloses a novel method to separate BBI and KI from crude plant extract which is cost effective and less laborious in shorter duration, using TCA at an early stage of protein extraction and purification.
Most plant tissues do not provide a ready source of proteins and thus need special extraction procedures. Due to the presence of the cell wall and the vacuole, protein extraction from plants is more difficult, because of proteolytic breakdown, streaking, and charge heterogeneity in plant cells. Most common interfering substances are phenolic compounds, proteolytic and oxidative enzymes, terpenes, pigments, organic acids, inhibitory ions, and carbohydrates. Thus, the development of efficient protein extraction methods for various plant tissues is important, yet very challenging.
Moreover, BBI and KI are even more difficult to separate because of their similar molecular masses and similar properties. The currently known methods for purifying BBIs and KIs from plants take several days, the method disclosed herein is more efficient, takes much less time compared to the known methods, and is also cost-effective.
Proteolytic enzymes, also called proteases, are the enzymes that catalyse the hydrolytic cleavage of specific peptide bonds in their target proteins. One important control mechanism for protease action involves interaction of the active enzymes with proteins that inhibit their activities. These inhibitors form less active or fully inactive complexes with their cognate enzymes, and are called protease or proteinase inhibitors (PIs).
Insect larvae feed on the vegetative and reproductive organs of plants and digest them with the aid of serine, cysteine, aspartic or metalloproteinases present in their gut environment. In response, plants mount many defence responses against pests and insects. From an economic perspective, Helicoverpa armigera (Ha) and Achaea janata (Aj) are relatively important among the lepidopteran insect pests. H. armigera, being polyphagous causes significant loss to many crops. Further, the management of H. armigera continues to be a major challenge as it has developed resistance to a variety of pesticides. Also, A. janata feeds on an oil-rich Ricinus communis and causes severe loss to this cash crop owing to its foliar feeding behaviour.
Plant serine proteinase/ protease inhibitors or Plant serine PIs (SPIs)are grouped into Kunitz (trypsin) inhibitors (KI or KTI), Bowman-Birk inhibitors (BBI), potato type I, and potato type II inhibitors, cereal trypsin/amylase, metallo-carboxypeptidase inhibitors, mustard trypsin inhibitor, cysteine protease inhibitors and squash inhibitor families.
Plant SPIs are of particular interest because they act as protective agents codified by a single gene and inhibit proteolytic enzymes from animal and fungi, but rarely from plants. The biotechnological potential of SPIs employed as protective agents has been demonstrated by transferring SPI genes from different sources to several plants of economic interest and the resulting transgenic plants are more resistant to pests and pathogens.
Plant SPIs can enter the insect digestive tract along with the plant food for the insects/ larvae, and block protein digestion, and the insect is unable to absorb the nutrients leading to the retardation of its growth and development. Thus, plant SPIs (soybean Kunitz and Bowman–Birk inhibitors) would get access to the proteases present in the insect gut. These BBI and KI proteins can thus help to control herbivorous insects.
PIs are a great contributor to plant defence and protection against all pathogenic organisms including pests, insects, microbes and herbivores. Standard protocols use ammonium sulphate fractionation followed by passing the protein through different columns (ion exchange, affinity and gel filtration) which is very time consuming, costly and laborious to purify seed proteins. Even after usage of this tedious protocol, separation of BBI and KI is not achieved due to the oligomeric nature of BBI and also the similar molecular mass of KI and BBI. The current methods of purifying PIs are performed by subjecting crude extract to ammonium sulphate fractionation and passing the sample through affinity and gel filtration chromatography columns, and these methods are highly laborious and time consuming.
Two major clusters of these inhibitors are KTIs and BBIs families. The main difference between their members is the number of disulfide linkages—BBIs contain usually seven, while most KTIs two disulfide bonds. KTIs contain a single reactive site, while, in some BBIs, there are two reactive sites. Moreover, both families share a similar mechanism of inhibition. They are found in legumes
Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal
grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motifCTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress.
Proteinase inhibitors (PIs) form stoichiometric complexes with specific proteolytic enzymes, thus preventing their catalytic function.
Kunitz type inhibitors (KTIs) or Kunitz inhibitors (KIs) are single chain polypeptides of~20 kDa, with two intra-chain disulphide bridges and form 1:1 complex with the target proteinase. The Bowman–Birk inhibitors (BBIs) are also single chain polypeptides, but smaller in size(~8 kDa) and possess seven disulphide bridges and two active domains for trypsin and/or chymotrypsin.
The term “Percentage yield recovery” as used herein defined as Protease inhibitor activity (in TI units) in a particular step divided by protease inhibitor activity (in TI units) of crude extract and multiplied with 100 give yield recovery.
The activity of PIs was expressed as trypsin inhibitor (TI) units/mg protein or chymotrypsin inhibitor (CI) units/mg protein. One TI or CI unit was defined as the amount of inhibitor required to inhibit 50% of the corresponding protease activity.
“AjGPI units (Achaea janata gut trypsin-like protease inhibitory activity)” represent the activity of BBI or KI in terms of its capacity to inhibit trypsin-like gut proteases present in the midgut of Achaea janata larva.
“HaGPI units (Helicoverpa armigera gut trypsin-like protease inhibitory activity )units represent the activity of BBI or KI in terms of its capacity to inhibit trypsin-like gut proteases present in the midgut of Helicoverpa armigera larva.
IC50 is the amount of BBI or KI that inhibits activity of the proteases by 50%.
Plant molecular farming is defined as a large-scale production of recombinant proteins for the purpose of their use as biotechnological products with various applications.
Embodiments:
One Embodiment of the current invention is a method of purifying protease inhibitor proteins Bowman-Birk inhibitor (BBI) and Kunitz inhibitor (KI) from plant seeds, the method comprising the steps of:
(a) making crude extract from plant seeds;
(b) extraction of the crude extract from step (a) with 2.5% Trichloro acetic acid (TCA);
followed by heating and centrifugation to obtain a first pellet fraction and a first supernatant fraction;
(c) performing acetone precipitation with the first supernatant fraction from the step (b) to obtain a second pellet fraction, dissolving the second pellet fraction in a buffer and performing affinity chromatography with the dissolved pellet to obtain BBI protein;
(d) dissolving the first pellet from step (b) in a buffer with pH 8-9 followed by TCA extraction, heating and centrifugation of the dissolved first pellet to obtain a double extracted third pellet fraction; and
(e) dissolving the third pellet fraction in buffer, and performing affinity chromatography of the dissolved third pellet fraction from step (d) followed by adding twice the volume of sodium acetate buffer, heating and centrifugation, subjecting supernatant to acetone precipitation to obtain a fourth pellet containing purified KI protein followed by dissolving the fourth pellet in a buffer to obtain dissolved KI protein.
In one embodiment, the crude extract in step (a) is made by crushing dried plant seeds, into fine powder, followed by depigmentation and defatting with repeated washes of acetone and hexane, respectively.
In one embodiment, the trichloroacetic acid (TCA) used for extraction in step b) is 2 to 3% TCA.
In one embodiment, the BBI and KI (PIs) are purified from plants of leguminosae (Fabaceae) , graminaceae and Solanaceae families. In one embodiment, the BBI and KI (PIs) are purified from plants of leguminosae family.
In one embodiment, the BBI and KI PIs are purified from leguminosae family members, examples of which include, but are not limited to, red gram, green gram, black gram, horse gram, soybean, chickpea and peanut.
In one embodiment, the BBI and KI PIs are purified from wild varieties of red gram varieties such as Rhynchosia sublobata and Cajanus platycarpus, and cultivated varieties of red gram.
In one embodiment, the yield of purified BBI using the method disclosed herein is 0.3 to 0.45mg protein per gram dried seeds, with 14 to 26 % yield recovery and 37-55 fold purification, and the yield of purified KI is 0.1 to 0.135 mg per gm seeds with 2.9 to 4% yield recovery and 25-fold purification), varied respectively.
In one embodiment, the purified BBI and KI proteins obtained by the method disclosed herein exhibit oligomerization (self association pattern). In one embodiment, the purified BBI and KI (PIs) have protease inhibitory activity against serine proteases (examples of which include, but are not limited to, trypsin and chymotrypsin).
In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable at pH 2.0 to 12.0. In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable at a temperature up to 100°C. In one embodiment, the BBI and KI PIs purified by the method disclosed herein are stable to reducing agents such as Dithiothreitol (DTT).
In one embodiment, the BBI PIs purified by the method disclosed herein exhibit specific activity against trypsin-like gut proteases of lepidopteran insect pest, Achaea janata (A. janata larval gut trypsin-like proteases ;AjGPs) (21,000 to 33,000 AjGPI units / mg protein with an IC50 of 22 to 96ng).
In one embodiment, the BBI PIs purified by the method disclosed herein cause significant weight reduction in larvae of A. janata by 76-83% of control larvae weight, mortality rates of larvae by 20-45% and showed inactive larval-pupal intermediates (30-50%) formation during its life cycle as compared with control larvae during their metamorphosis from larva to pupa stage.
In one embodiment, the KI PIs purified by the method disclosed herein exhibit specific activity against trypsin-like gut proteases of lepidopteran insect pest Helicoverpa armigera (18,520 to 26,240 HaGPI units/mg protein with an IC50 of 42 to 150 ng).
In one embodiment, the KI PIs purified by the method disclosed herein lead to significant weight reduction in H. armigera larvae by 66-70% of control larvae weight, mortality rates of larvae by 20-33% and showed inactive larval-pupal intermediates (36-52%) formation during its life cycle as compared with control larvae during their metamorphosis from larva to pupa stage.
In one embodiment, the BBI and KI PIs purified by the method disclosed herein (PI) exhibit 33% reduction in the growth of epithelial breast cancer cell lines (MCF-7) as compared with control (MCF-10A) non-cancerous cell lines
In one embodiment, the BBI and KI PIs purified by the method disclosed herein lead to 60% inhibition in the growth of methicillin-sensitive Staphylococcus aureus bacteria cells as compared with control cells (without KI/PI).
In one embodiment, the method disclosed herein is used for purifying KI and BBI protein from seeds of the plant family Leguminosae,
In one embodiment, the method disclosed herein is used for purifying KI and BBI protein from seeds of the plant family Leguminosae.
In one embodiment, the method further comprises the step of estimation of protein concentration and purity of the crude extract, pellet fraction and supernatant from step (b), double extracted pellet fraction from step (d), and the purified BBI and KI proteins from steps (c) and (e) by the above disclosed method. Protein quantitation may be used for determination of protein concentration at any of the separation or purification steps in the above disclosed method.
In one embodiment, protein concentration can be quantitated by any of the known methods, including but not limited to BCA kit method.
In one embodiment, the method further comprises the steps of determining purity, stability and activity of the purified BBI and KI proteins.
In one embodiment, the method disclosed herein is used for isolating purified BBI and KI PIs from any member of the leguminosae family. In one embodiment, the legume plant can be, but is not restricted to red gram, black gram, green gram, horse gram, chickpea and peanut.
In one embodiment, the method disclosed herein is used for isolating purified BBI and KI PIs from R. sublobata plants.
R. sublobata is a wild relative of pigeon pea, containing both BBI and KI type of PIs.
In one embodiment, the method disclosed herein is used for isolating purified BBI and KI PIs from plants of graminaceae family. In one embodiment, the graminaceae family plants include rice, wheat, or other cereal plants.
In one embodiment, the method disclosed herein is used for isolating purified BBI and KI PIs from plants of Solanaceae family. In one embodiment, the Solanaceae family plant is tomato, eggplant, pepper, or potato plants.
In one embodiment, the KI and BBI proteins purified by the current method have insecticidal/ pesticidal activity. In one embodiment, the plant pests belong to the order Lepidoptera or other orders of class Insecta and phylum Arthropoda.
In one embodiment, the KI and BBI proteins purified by the current method have insecticidal/ pesticidal activity and act as defence proteins against pests and pathogens.
In one embodiment, the KI and BBI proteins purified by the current method have apoptosis modulatory activity.
In one embodiment, the KI and BBI proteins purified by the current method possess protease inhibitory activity – mostly against serine proteases (e.g. trypsin, chymotrypsin, elastase, metallo-proteases, Cathepsin G etc.)
In one embodiment, the KI and BBI proteins purified by the current method can be included in Protease inhibitor cocktail.
In one embodiment, the KI and BBI proteins purified by the current method can be used as a protein marker. In one embodiment, the KI and BBI proteins purified by the current method can be used as preservatives. In one embodiment, the KI and BBI proteins purified by the current method have antifungal properties. In one embodiment, the KI and BBI proteins purified by the current method have antimicrobial properties.
In one embodiment, the KI and BBI proteins purified by the current method have antiproliferative activity against tumour cells, and have anti-tumour properties. In one embodiment, the KI and BBI proteins purified by the current method are used in cancer therapy and as chemo preventive agent. In one embodiment, the KI and BBI proteins purified by the current method have antiviral properties
In one embodiment, the KI and BBI proteins purified by the current method have anti-HIV properties. In one embodiment, the KI and BBI proteins purified by the current method have anti-malarial properties.
In one embodiment, the KI and BBI proteins purified by the current method have therapeutic, pharmaceutical and medicinal properties
In one embodiment, the KI and BBI proteins purified by the current method can be used in molecular farming during biotechnological applications, for producing recombinant proteins. In one embodiment, the KI and BBI proteins purified by the current method can be used as an affinity adsorbent to purify proteases.
In one embodiment, the method disclosed herein takes approximately two days for purification of BBI and KI proteins. In one embodiment, the BBI proteins purified by the method disclosed herein are stable at 100°C. In one embodiment, the KI proteins purified by the method disclosed herein are stable at 70°C.
In one embodiment, the BBI and KI proteins purified by the method disclosed herein are stable at pH 2.0 to 12.0. In one embodiment, the KI proteins purified by the method disclosed herein are stable against reducing agents such as DTT.
In one embodiment, the different isoforms of KI and BBI can be isolated by the method disclosed herein.
In one embodiment, the BBI and KI isolated by the method disclosed herein can get oligomerized.
In one embodiment, the crude extract in step (a) of the method is made by taking mature dried seeds that are crushed into fine powder, depigmented and defatted with repeated washes of acetone and hexane, respectively.
In one embodiment, the trichloroacetic acid (TCA) used for extraction is 2 to 3 % TCA. In one embodiment, the TCA is 2.5 %.

EXAMPLES
Example 1: Purification and separation of BBI and KI:
Materials and methods:
Mature dry seeds from Rhynchosia sublobata (Rs), Cajanus Platycarpus (Cp), Green gram (Gg)(10g) were crushed into fine powder, depigmented and defatted with two/three washes of acetone and hexane, respectively. The powder obtained was air dried and extracted with (1:6 w/v) 50 mM Tris-HCl (pH 8.0) containing 1% polyvinyl pyrrolidine with constant stirring for 3 hrs at 4°C. The extract was centrifuged at 12,000 rpm for 15 min at 4°C and the supernatant was collected. The crude extract containing 40 mg/ml protein was subjected to trichloroacetic acid (TCA) extraction (2.5%) and heated at 70°Cfor10 min followed by centrifugation at 12,000 rpm at room temperature (RT) for 5 min. The supernatant containing TCA soluble BBI was collected (S1a) and adjusted to pH 8.0 with 400 mM Tris-HCl and acetone precipitated by mixing at 1:4 ratio (v/v). The pellet (P2) obtained with TCA from crude extract was again subjected to TCA extraction and the supernatant (S1b) containing any left over BBI was collected and acetone precipitated by mixing at 1:4 ratio (v/v). Both the acetone precipitated samples from S1a and S1b are centrifuged at 12,000 RPM for 15 min (4°C). The protein pellets obtained after centrifugation are dissolved in 50 mM Tris-HCl (pH 8.0) and pooled, and applied on to Trypsin sepharose4B column which is pre-equilibrated with 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl. The proteins that were unbound to the column are washed out and the bound proteins were eluted with0.01N HCl. The fractions (1.0 mL) showing trypsin inhibitory activity were pooled up, concentrated using amicon filters (3kDa) (Millipore) and stored at-20 o C as pure BBI until further use.
The pellet (P3) obtained after second time TCA extraction was dissolved in 50 mM Tris HCl (pH 8.0) and applied on to Trypsin sepharose4B column (GE healthcare). The fractions showing trypsin inhibitory activity were pooled up, concentrated using amicon filters (3kDa). Further, the KI present in the affinity fractions was extracted into 100 mM sodium acetate buffer (pH 4.0) by mixing at 1:2 ratios (v/v), and heated at 70°Cfor10 min before centrifuging at 12,000 rpm at RT for 5 min. The supernatant (S2) was collected and adjusted to (pH 8.0) with 100 mM Tris HCl and acetone precipitated by mixing at 1:4 ratio (v/v). The protein pellet (P4) obtained after centrifugation at 12,000 RPM (4°C) is air dried and dissolved in 50 mM Tris- HCl (pH 8.0), and labeled as pure KI, and stored at -20°C.
Results:
Protein purification is vital for analyzing its function, structure and interaction with other molecules. Purification of a single protein from a complex mixture of other protein and non-protein components involves a series of steps right from preparation of crude extract to passing through different chromatography columns which is very laborious and time consuming. Plant tissues have a relatively low protein content compared to bacterial or animal tissues and need special precautions to obtain pure protein. Most common interfering substances in protein purification are phenolic compounds, proteolytic and oxidative enzymes, terpenes, pigments, organic acids, inhibitory ions, and carbohydrates. Thus, the development of efficient protein extraction methods for various tissues is important.
Proteinase inhibitors (PIs) are small defence molecules which act as natural antagonists of proteolytic enzymes (Marathe et al., 2019, ref 8). Based on the type of proteases they inhibit, they are classified into serine, cysteine, aspartic and metallo PIs. Among them serine PIs are well studied type of PIs which include Bowman Birk (BBI) and Kunitz inhibitors (KI) . The information on identification of BBI and KI from same seed variety is very limited and reported from very limited number of seed varieties such as Glycine max, Clitoriaternatea, Cajanus cajan and Mucuna pruriens so far (Catsimpoolas 1969(ref 2), Kumar et al., 2018 (Ref 5), Roosta et al., 2011 (Ref 14), Xavier-Filho and Macedo 1962(Ref 7)). Besides, both BBI and KI are collected into the same fractions during various purification steps. Various protocols were used by several research groups to separate BBI and KI based on isoelectric focusing, solvent extraction, gel elution and passing through gel filtration, affinity and ion exchange columns. However, a standard protocol for separation of these two PIs is not yet standardised. Moreover, separation of BBI and KI from single seed by using the above mentioned chromatography techniques is highly difficult as they share some common features such as i) both are trypsin inhibitors ii) exist as oligomers with close molecular mass iii) exist as isoforms with overlapping isoelectric points.
According to Mohanraj et al. (2019)(Ref 9) even after passing through different columns, BBI and KI are inseperable and required additional steps such as extracting with TCA and sodium acetate to obtain pure BBI and KI fractions. TCA is an effective protein precipitating agent which increases their hydrophobicity by exposing the hydrophobic core of proteins (Novak and Havlicek 2016) (Ref 10). BBI is a small, globular protein with hydrophilic core and it is assumed to be in the supernatant whereas KI with hydrophobic core is assumed to precipitate upon TCA extraction. Therefore, in the present study, a new protocol was applied to separate BBI and KI from mature dry seeds of legume plants [Green gram (Vigna radiata), red gram wild relatives such as Cajanus platycarpus (ICPW-68) and Rhynchosia sublobata] by subjecting to TCA extraction from crude extract itself (Fig. 1). Initially, BBI was separated from crude extract by subjecting to TCA extraction and heating at 70°C (step repeated).It is anticipated that heating will denature the heat labile proteins after TCA extraction and the BBI which is soluble in TCA is stable even upto boiling temperature (100°C) due to presence of seven conserved disulfide bridges. The BBI collected into supernatant (S1a& S1b) is acetone precipitated, air dried and the protein pellets obtained are subjected to trypsin affinity chromatography after dissolving in 50 mM Tris-HCl at pH 8.0 (Figure 1). The BBI bound to the trypsin-sepharose column was eluted with 0.01N HCl. The second peak (Fig. 2A) showing TI activity was concentrated using amicon filters (3kDa) and labelled as ‘BBI’. The pellet ‘P3’ fraction obtained after second time TCA extraction, which is enriched with KI is subjected to trypsin affinity chromatography after dissolving in 50 mM Tris- HCl at pH 8.0 (Figure 1).The protein is eluted with 0.01N HCl and the second peak showing TI activity in chromatogram was concentrated (Fig. 2B) using amicon filters (3kDa), and extracted with sodium acetate to obtain ‘KI’. This protocol resulted in approximately0.35 to 0.4 mg of pure BBI and 0.1 to 0.2 mg of pure KI per gm seed of different Leguminosae varieties chosen in the present study (Table.1). The purity obtained after each step was visualized in Tricine SDS PAGE (15%) under non-reducing conditions (Fig.2C, D, and E).
Table. 1. Purification table showing the purification fold, specific activity and yield recovery of BBI and KI separated from mature dry seeds (10g) of green gram, Cajanus platycarpus (ICPW 68) and R. sublobata.
Purification step Total protein (mg) Total activity (TI units) Yield Recovery (%) Specific Activity (TI units/mg protein) Purification (fold)
Green gram
Crude extract 858 25588 100 30 1
TCA extraction 235 12525 49 53.2 1.8
Pure BBI 3.5 4584 18 1309 44
Pure KI 1.34 1042 4 778 26
C. platycarpus
Crude extract 900 34587 100 38.4 1
TCA extraction 340 16321 27 48 1.25
Pure BBI 3.5 4946 14.3 1413 37
Pure KI 1.0 980 2.83 980 25.5
R. sublobata
Crude extract 954 34496 100 36.15 1
TCA extraction 304 22858 66.3 75.2 2.08
Pure BBI 4.50 8912 26.0 1980.44 54.8
Pure KI 1.20 1046 3.03 886 24.5

Example 2: Biochemical characterizationof BBI and KI
2.1 Estimation of protein and inhibition assay:
Protein concentration of crude extract, TCA extracted fractions, purified BBI and KI fractions was estimated by Bicinchoninic acid (BCA) kit method using BSA as a standard. The inhibitory activity of BBI and KI against different serine proteases (trypsin and chymotrypsin) was determined by monitoring the reduction in the activity of these proteases. For this, the assay mixture containing PIs in assay buffer (50 mM Tris and 20 mM CaCl2 pH 8.2 for trypsin and pH 7.8 for chymotrypsin) were incubated with respective proteases (trypsin and chymotrypsin) for 15 min at 37 °C. The residual protease activity of the assay mixture was determined after incubating with substrates N-a-benzoyl-DL-arginine-p-nitroanilide (BAPNA) for trypsin and N-glutaryl
-L-phenylalanine-p-nitroanilide (GLUPHEPA) for chymotrypsin for 45 min at 37 °C. The reaction was terminated by adding 30% acetic acid and absorbance at 410 nm was recorded. The activity of PIs was expressed as trypsin inhibitor (TI) units/mg protein or chymotrypsin inhibitor (CI) units/mg protein. One TI or CI unit was defined as the amount of inhibitor required to inhibit 50% of the corresponding protease activity.
Example 2.2: Electrophoretic separation of BBI and KI
Tricine-SDS-PAGE was performed using 4% stacking gel and 15% separating gel to check the purity of BBI and KI. Further, the gel was stained by using silver nitrate method (Prasad et al., 2010) (Ref 12). The fractions obtained from each purification steps were analyzed electrophoretically along with protein molecular marker (10 to 100 kDa) and commercial soybean BBI (8.0 kDa) dissolved in 50 mM Tris-HCl, (pH 8.0) to avoid the variation in molecular mass and mobility of BBIs with other proteins (Swathi et al., 2016)(Ref 16).
Example 2.3: MALDI Intact Mass analysis
Protein mass of the purified proteins was determined by mass spectroscopic (MALDI-TOF orESI-Q-TOF)method. The sample was diluted with 1% Formic acid and it was analyzed on the ESI-Q-TOF instrument for intact molecular weight by direct infusion. The raw data obtained was processed and deconvoluted by MassLynx 4.1 WATERS software. In contrast, the molecular mass of protein in MALDI-TOF was determined by mixing the sample with a-cyano-4- hydroxy-cinnamic acid matrix and the spectra obtained were analyzed using Flex analysis version 3.1 software.

Example 2.4: Visualization of in gel activity: For in-gel activity staining studies, the gels were incorporated with gelatin. After electrophoresis, the gels were washed thrice with triton X-100 for 15 min followed by incubation for 1 h at 37°C with respective proteases dissolved in 0.1 M Tris–HCl, pH 8.2 (Trypsin/AjGPs), 0.1 M Tris–HCl, pH 7.8 (Chymotrypsin) or 0.1 M glycine–HCl, pH 10.5 (HaGPs) buffer solutions, respectively. The inhibitory bands were visualized in blue on a white background after staining with Coomassie brilliant blue R-250.
Example 2.5: Stability studies
The effect of temperature on inhibitory activity of BBI and KI was tested by incubating at 37°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°Cand100°Cfor 30 min. After cooling the samples to RT the residual trypsin and chymotrypsin inhibitory activity was assayed at 37°Cin presence of the substrates BAPNA and GLUPHEPA, respectively. The impact of pH on trypsin and chymotrypsin inhibitory activity of BBI was examined at pH ranging from 2 to 12 using the following buffers at final concentrations of 50 mM: glycine-HCl (pH 2-3), sodium acetate-acetic acid(pH 4-5), sodium phosphate buffer (pH 6.0), Tris-HCl (pH 7-9) and glycine-NaOH (pH10-12).After pre-incubation at 37°C for 1 h in the respective buffers, the residual inhibitory activity was measured at pH 8.2 for trypsin and pH 7.8 for chymotrypsin. Also the stability in TI/CI activities of BBI and KI against DTT was determined by incubating at different concentrations of DTT i.e. up to 3 mM with BBI and 200 mM with KI at 56°C for 45 min followed by incubating with iodoacetamide at double the concentration of DTT in dark for 1 h. The residual inhibitory activity against trypsin and chymotrypsin was estimated using BAPNA and GLUPHEPA as substrates.
One third of all known proteolytic enzymes are serine proteases which are widely distributed in all kingdoms of cellular life (Di cera 2009 (Ref 3). As BBI and KI are serine PIs, the effect of BBI and KI on two serine proteases such as trypsin and chymotrypsin was examined by in vitro inhibition assay and in-gel activity staining studies. BBIs showed 1309-1980 TI units/mg protein and 240-460 CI units/mg protein where as KIs showed 778-980 TI units/mg protein, but not showed any inhibition against chymotrypsin (Fig. 3A, B) which was in correlation with in-gel activity staining studies (Fig. 3C, D). Loading of increased concentrations of BBIs and KIs showed oligomers in Tricine SDS-PAGE (Fig. 4A), which was further evident during MALDI intact mass analysis (Figs. 4B-E). The molecular masses of all BBI and KI purified in the present study using mass spectrometry are shown in Figure 4F and Table 2. MALDI Q-TOF analysis of the purified BBIs showed a predominant peak between 7-9 kDa as a monomeric peak and small peak at 15-18 kDa as a dimeric peak and other higher-ordered oligomeric forms such as trimer, tetramer, pentamer and hexamer between 5000-60,000 m/z (Fig. 4B,D). For KIs, molecular masses at 19 kDa (monomer), 38 kDa (dimer) and 57 kDa (trimer) was observed (Fig. 4C,E). The MALDI Intact mass analysis of CpBBI and GgKI showed different isoforms as well as higher ordered oligoforms (insets of Fig. 4D, E). The low molecular weight, self association pattern and presence of trypsin and chymotrypsin activity were in agreement with BBIs purified from other seed varieties such as peanut, black gram and red gram (Lokya 2020(Ref 6), Prasad et al., 2010 (Ref 12), Swathi et al., 2016 (Ref 16)).
Table 2: Molecular masses of all BBI and KI purified in the present study using mass spectrometry (also shown in Fig. 4F)
Protein Molecular weight
GgBBI 8.2 kDa
CpBBI 7.9 kDa
RsBBI 9.2 kDa
GgKI 19.2 kDa
CpKI 19.4 kDa
RsKI 19.3 kDa

PIs are known to have high cysteine content which offers them greater stability. Hence the effect of temperature, pH as well as reduction of cysteine residues with DTT on the activity of these PIs was examined by performing inhibition assays. Inhibitory activity of BBI against trypsin and chymotrypsin was examined after incubating at different temperature conditions for 30 min. Trypsin inhibitory activity of BBI was stable until 30 min even after heating up to 90°C. However, marginal loss in trypsin and chymotrypsin inhibitory activity (5-15%) was observed when heated for 100°C (Fig. 5A, B). Conversely, TI activity of KI was stable up to 70°C but lost its activity when heated to 100°C (Fig. 5C). The inhibitory activity of BBI against trypsin and chymotrypsin was stable at different pH conditions tested between 2.0 and 12.0 (Fig. 5D, E). Except at pH 6.0, only negligible loss in trypsin inhibitory activity was observed for KIs also (Fig. 5F). The loss (30-40%) in inhibitory activity at pH 6.0 could be due to isoelectric precipitation at that particular pH. Disulfide bonds help in maintaining the structural conformation and functional stability of a protein against various pH and temperature treatments. Reduction of disulfide bonds by using reducing agent resulted in significant loss of activity against trypsin and chymotrypsin of BBI (Fig. 5G, H). At 0.25 mM concentration of DTT there was a significant decrease in the inhibitory activity of BBI against trypsin and chymotrypsin (80%). By increasing the concentration of DTT to 1.0 mM, there was a complete loss in inhibitory activity against trypsin and chymotrypsin. The TI activity of KI was highly stable to the action of DTT. It lost upto50% of its activity at 25 mM, but lost its activity completely at a concentration of 200 mM (Fig. 5I).
Example 3: Extraction of midgut proteases from Achaea janata and Helicoverpa armigera:
The midgut from fifth instar larvae of control insects (obtained from National Bureau of Agricultural Insect Resources (NBAIR), Bangalore) was dissected by narcotizing them on ice for 45 min using iso-osmotic saline and stored at -80°C until further use. The gut tissue was homogenized with a glass homogenizer by adding 0.1 M Glycine-NaOH (pH 10.5) for H. armigera and 0.1 M Tris HCl (pH 8.2) for A. janata followed by centrifugation at 12,000 rpm for 10 min at 4°C. The obtained clear supernatant enriched with AjGPs and HaGPs was used for in vitro enzyme inhibition assays and in-gel activity staining studies as mentioned in 2.2 and 2.5, respectively.
3.1 In vitro inhibitory activity of BBI and KI against HaGPS and AjGPs (HaGPs: Helicoverpa armigera gut proteases; AjGPs: Achaea janata gut proteases):
Proteases are the essential enzymes in insects as they hydrolyze the proteins and release amino acids that are necessary for their growth and development. Majority of the proteases in Lepidopteran insect gut belongs to serine type such as trypsin, chymotrypsin, elastase and few others such as aspartic, metallo and cysteine proteases (Patankar et al., 2001 (Ref 11), Srinivasan et al., 2006 (Ref 15), Tabatabaei et al. 2011 (Ref 17). Therefore, the inhibition of these midgut proteases by BBIs and KIs was tested by in vitro inhibitory assays and in-gel activity staining studies. The specific activity of BBIs and KIs against Achaea janata gut proteases (AjGPs) and Helicoverpa armigera gut proteases (HaGPs) showed broad range of differences. Specific activity of BBIs (21,200 to 33,940 AjGPI units/mg protein) and KIs(478 to 1548AjGPI units/mg protein) against AjGPs varied greatly (Fig. 6A). In contrast, the specific activity of BBIs (215 to 922HaGPI units/mg protein) against HaGPs was less as compared to KIs (18,520 to 26,240 HaGPI units/mg protein) (Fig. 6B). These results demonstrate that BBIs purified from these seed varieties are highly specific and strong inhibitors of AjGPs whereas KIs are specific towards HaGPs. Besides, these results were confirmed by in-gel activity staining studies (Fig. 6C, D). Further, BBIs exhibited strong inhibitory potential against AjGPs with an IC50 of 20 to 100 ng as compared to KIs (IC50 of 2.5 to 4.6 µg) (Table, Fig.7A,B,C). Conversely, KIs inhibited HaGPs to an extent of 80% with an IC50 of 40 to 150 ng as compared to BBIs which showed an IC50 of 4.2 to 7.8 µg (Fig. 7D,E,F).
Table 3: Inhibitory potential of BBI and KI against midgut proteases of A. janata and H. armigera. Half-maximal inhibitory concentration (IC50) of BBIs and KIs against AjGPs (Fig. 7C)
Protein IC50 against AjGPs
GgBBI 0.096 µg
CpBBI 0.084 µg
RsBBI 0.022 µg
GgKI 4.2 µg
CpKI 2.6 µg
RsKI 3.5 µg

Table 4: Inhibitory potential of BBI and KI against midgut proteases of A. janata and H. armigera. Half-maximal inhibitory concentration (IC50) of BBIs and KIs against HaGPs (Fig. 7F)
Protein IC50 against HaGPs
GgBBI 6.0 µg
CpBBI 4.2 µg
RsBBI 7.5 µg
GgKI 0.15 µg
CpKI 0.080 µg
RsKI 0.042 µg

Example 4: Effect of BBI and KI on larval growth and development of A. janata and H. armigera
The newly hatched larvae on castor leaves were maintained at temperature (26 ± 1oC), relative humidity (65 ± 5%) and photoperiod (14:10 h) in insect culture room. The effect of BBI and KI on growth and development of A. janata was assessed by feeding newly-hatched larvae on castor leaves coated with BBI and KI at 8 µg/cm2. Control leaves were coated with 50 mM Tris-HCl (pH 8.0). Whereas the effect of BBI and KI on the larval growth and development of H. armigera was assessed by feeding newly-hatched larvae with BBI or KI supplemented artificial diet at concentration of 0.05%. Control larvae were fed on artificial diet without BBI or KI. The weight of control and treated larvae was recorded before they showed metamorphosis into a pupa. Reduction in larval growth and formation of larval-pupal intermediates was recorded by taking weights and photographic images.
4.1. In vivo effect of BBI and KI on growth and development of A. janata: Achaea janta, the castor semi looper, is a major pest of castor throughout the world. BBIs and KIs showed wide differences in controlling growth and development of A. janata in terms of larval weight, pupal formation and survival rate. Significant reduction in the body weight (76-83.4%) was observed at 5th instar stage of larvae upon feeding the castor leaves coated with 8 µg/cm2 concentration of BBIs purified from green gram, C. platycarpus and R. sublobata, respectively when compared with larvae fed on control leaves (Fig. 8A, B). Feeding of BBIs also resulted in reduction in pupal weights, delayed pupal emergence (10-12 days), formation of larval-pupal intermediates (30-50%) and about 10-35% mortality was recorded (Fig. 8C, D,E). Whereas KI (8 µg/cm2) fed larvae showed mild reduction in larval body weight (10-30%) as compared to BBI fed larvae. Though no mortality was observed, however, showed formation of larval-pupal intermediates (10 to 30%) (Fig. 8D,E). The observed mortality and arrested growth at larval and pupal stage could be due to the starvation-induced stress on the expression of gut proteases.
4.2 In vivo effect of BBI and KI on growth and development of H. armigera:
H. armigera is a polyphagous pest of several economically important crops such as tomato, cotton, corn, tobacco, groundnut, chilli, bhendi, pigeon pea and chickpea (Talekar et al., 2006 (Ref 18), Elumalai et al., 2010 (Ref 4), Rauf et al., 2019 (Ref 13). The antifeedant activity of BBI and KI on larval growth and development was tested by feeding the first instar larvae of H. armigera on 0.05% BBI or KI supplemented artificial diet. The 5th instar larvae showed 66-70% of reduction in the body mass when fed on diet mixed with 0.05% KI compared with control insects (Fig. 9A & B). Similarly, the pupal weights of the respective larvae fed on KI (0.05%) also decreased by 53-56% when compared to their controls (Fig. 9C). The 0.05% of KI fed larvae showed significant results such as 10-14 days of delay in pupal emergence, formation of larval-pupal intermediates (Fig. 9D & E) and 20-32% mortality (Fig. 9E). Whereas BBI showed 10-20% reduction in larval weight and the formation of larval pupal intermediates was very less (8-12%) when compared with insects fed on KI, indicating the specificity of BBI and KI against different Lepidopteran insects (Fig. 9A,B, D).
Table 5 and 6: Effect of BBI and KI on growth and development of A. Janata (Fig. 8E and 9E respectively )

Protein
(8 µg/cm2) Mortality rate
(% control) Larval-pupal intermediates (% control)
GgBBI 20 40
CpBBI 35 50
RsBBI 45 30
GgKI 0 10
CpKI 0 30
RsKI 0 No

Table 6
Protein
(0.05%) Mortality rate
(% control) Larval-pupal intermediates (% control)
GgBBI 0 8
CpBBI 0 12
RsBBI 0 No
GgKI 20 40
CpKI 28 52
RsKI 32 36

Example 5: Therapeutic properties of purified KIs
5.1: Anti-cancer activity of Purified KIs: The anticancer effects of purified KIs from various seeds was evaluated on 3 cancer cell lines (MCF7, MDA-MB-231, HeLa) and a normal non-cancerous cell line (MCF10A). MCF7, MDA-MB-231 are cancerous breast epithelial cells, HeLa cells are cancerous cervical epithelial cells and MCF10A are normal non-cancerous breast epithelial cells. All the cell lines were obtained from National Center for Cell Sciences (NCCS) Pune, India. The commercially available soybean BBI (from Sigma-Aldrich) was used as standard in all the experiments. The cytotoxicity MTT assay was performed as described earlier (Arunasree, 2010)(Ref 1). After preliminary experiments, final concentration of 10 µg of each purified KI/BBI was used in the MTT assay.
The anti-cancer effects of purified KIs from R. sublobata seeds were evaluated on two breast cell lines (MCF7, MDA-MB-231) and one cervical cancer cell line (HeLa). RsKI showed inhibition in the growth of cancer cell lines in the following order: epithelial breast cancer cell line MCF7 (33%) > MDA-MB-231 epithelial breast cancer cell line (14%) > Cervical cancer HeLA cell lines (7.5%) as compared to non-cancerous cell lines (MCF10A), respectively, which suggest that KIs have potential targets in breast cancer cells as compared with cervical cancer cells (Fig. 10A).
5.2: Anti-bacterial activity of purified KIs:
The antibacterial effects of purified KIs from various seeds were evaluated using Methicillin-sensitive Staphylococcus aureus (MSSA). MSSA (ATCC 29213) was obtained from ATCC. The antibacterial activity was evaluated using broth micro-dilution method as per CLSI guidelines (Clinical and Laboratory Standards Institute [CLSI], 2012). Briefly, 1% of the overnight grown MSSA culture in LB broth was inoculated into 10 ml of fresh LB medium and grown till log phase. The bacterial cells were then diluted to 50 McFarlands as per the CLSI protocol and 100 µl of cells were seeded into a 96-well plate. Different concentrations of the purified proteins were added into the wells and the total volume of the wells was made upto 200 µl with LB media and incubated for 12-16 hrs at 37oC. After incubation, 20 µl of 5 mg/ml MTT was added and incubated for another 3 hrs at 37oC. The formazan crystals formed were dissolved in 50 µl of DMSO and the absorbance was read at 595 nm. The % Growth was calculated as a fraction of control cells without the drug.
The antibacterial effects of purified KIs from seeds used in this method were evaluated using Methicillin-sensitive Staphylococcus aureus (MSSA), gram-positive bacteria. CpKI and GgKI showed concentration (2 to 10 ng/µl) dependent inhibition in the growth of bacteria, however, the optimal concentration for inhibition of growth varied between different KIs. CpKI showed maximum inhibition in the growth at a concentration of 4ng/µl whereas GgKI showed maximum inhibition in the growth at a concentration of 8ng/µl (Fig. 10B).
2.11. Statistical analysis:The data shown is Mean ± SE/SD of three biological replicates. Statistical differences were determined by one-way ANOVA analysis followed by Tukey test at a significance level of P = 0.05 using Sigma plot, version 12.0, Systat Software Inc., San Jose, CA, United States.
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,CLAIMS:1. A method of purifying protease inhibitor proteins Bowman-Birk inhibitor (BBI) and Kunitz inhibitor (KI) from plant seeds, the method comprising the steps of:
(a) making crude extract from plant seeds;
(b) extraction of the crude extract from step (a) with 2.5% Trichloro acetic acid (TCA);
followed by heating and centrifugation to obtain a first pellet fraction and a first supernatant fraction;
(c) performing acetone precipitation with the first supernatant fraction from the step (b) to obtain a second pellet fraction, dissolving the second pellet fraction in a buffer and performing affinity chromatography with the dissolved pellet to obtain BBI protein;
(d) dissolving the first pellet from step (b) in a buffer with pH 8-9 followed by TCA extraction, heating and centrifugation of the dissolved pellet to obtain a double extracted third pellet fraction; and
(e) dissolving the third pellet fraction in buffer, and performing affinity chromatography of the dissolved third pellet fraction from step (d) followed by adding twice the volume of sodium acetate buffer, heating and centrifugation, subjecting supernatant to acetone precipitation to obtain a fourth pellet containing purified KI protein followed by dissolving the fourth pellet in a buffer to obtain dissolved KI protein.
2.The method as claimed in claim 1, wherein the crude extract in step (a) is made by crushing dried plant seeds into fine powder, followed by depigmentation and defatting with repeated washes of acetone and hexane, respectively.
3.The method as claimed in claim 1, wherein the trichloroacetic acid (TCA) used for extraction in step b) is 2 to 3 % TCA.
4. The method as claimed in claim 1, wherein the BBI and KI proteins are purified from plants of leguminosae , graminaceae or Solanaceae plant families.
5. The method as claimed in claim 1, wherein the BBI and KI proteins are purified from plants of leguminosae family.
6.The method as claimed in claim 5, wherein the BBI and KI proteins are purified fromseeds of red gram, green gram, black gram, horse gram, soybean, chickpea or peanut plants.
7. The method as claimed in claim 1, wherein the yield of purified BBI protein is0.3 to 0.45mg protein per gram dried seeds and yield recovery is 14 to 26 % and the protein is purified 37-55 fold from total seed protein.
8. The method as claimed in claim 1, wherein the yield of purified KI protein is 0.1 to 0.135 mg per gm seeds and yield recovery is 2.9 to 4% and the KI protein is purified ~25-fold.
9. Purified BBI and KI proteins obtained by the method as claimed in claim 1.
10. The method as claimed in claim 1, wherein the purified BBI and KI proteins are stable at pH 2.0 to 12.0.
11. The method as claimed in claim 1, wherein the purified BBI protein is stable at a temperature up to 100°C.
12. The method as claimed in claim 1, wherein the separated and purified KI protein is stable to reducing agents such as Dithiothreitol (DTT).
13. The method as claimed in claim 1, wherein the purified BBI protein exhibits activity against trypsin-like gut proteases of lepidopteran insect pest, Achaea janata with an IC50 of 22 to 96ng.

14. The method as claimed in claim 1, wherein the purified KI protein exhibits activity against trypsin-like gut proteases of lepidopteran insect pest Helicoverpa armigera with an IC50 of 42 to 150 ng.