Bowman-Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities.

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Protease Inhibitors from Plants as Therapeutic Agents- A Review.

Plant-based diets are a great source of protease inhibitors (PIs). Two of the most well-known families of PIs are Bowman-Birk inhibitors (BBI) and Kunitz-type inhibitors (KTI). The first group acts mainly on trypsin, chymotrypsin, and elastase; the second is on serine, cysteine, and aspartic proteases. PIs can retard or inhibit the catalytic action of enzymes; therefore, they are considered non-nutritional compounds; nevertheless, animal studies and cell line experiments showed promising results of PIs in treating human illnesses such as obesity, cardiovascular diseases, autoimmune diseases, inflammatory processes, and different types of cancer (gastric, colorectal, breast, and lung cancer). Anticarcinogenic activity's proposed mechanisms of action comprise several inhibitory effects at different molecular levels, i.e., transcription, post-transcription, translation, post-translation, and secretion of cancer cells. This work reviews the potential therapeutic applications of PIs as anticarcinogenic and anti-inflammatory agents in human diseases and the mechanisms by which they exert these effects.

Extensive structural variation in the Bowman-Birk inhibitor family in common wheat (Triticum aestivum L.).

BACKGROUND: Bowman-Birk inhibitors (BBI) are a family of serine-type protease inhibitors that modulate endogenous plant proteolytic activities during different phases of development. They also inhibit exogenous proteases as a component of plant defense mechanisms, and their overexpression can confer resistance to phytophagous herbivores and multiple fungal and bacterial pathogens. Dicot BBIs are multifunctional, with a "double-headed" structure containing two separate inhibitory loops that can bind and inhibit trypsin and chymotrypsin proteases simultaneously. By contrast, monocot BBIs have a non-functional chymotrypsin inhibitory loop, although they have undergone internal duplication events giving rise to proteins with multiple BBI domains. RESULTS: We used a Hidden Markov Model (HMM) profile-based search to identify 57 BBI genes in the common wheat (Triticum aestivum L.) genome. The BBI genes are unevenly distributed, with large gene clusters in the telomeric regions of homoeologous group 1 and 3 chromosomes that likely arose through a series of tandem gene duplication events. The genomes of wheat progenitors also contain contiguous clusters of BBI genes, suggesting this family underwent expansion before the domestication of common wheat. However, the BBI gene family varied in size among different cultivars, showing this family remains dynamic. Because of these expansions, the BBI gene family is larger in wheat than other monocots such as maize, rice and Brachypodium. We found BBI proteins in common wheat with intragenic homologous duplications of cysteine-rich functional domains, including one protein with four functional BBI domains. This diversification may expand the spectrum of target substrates. Expression profiling suggests that some wheat BBI proteins may be involved in regulating endogenous proteases during grain development, while others were induced in response to biotic and abiotic stresses, suggesting a role in plant defense. CONCLUSIONS: Genome-wide characterization reveals that the BBI gene family in wheat is subject to a high rate of homologous tandem duplication and deletion events, giving rise to a diverse set of encoded proteins. This information will facilitate the functional characterization of individual wheat BBI genes to determine their role in wheat development and stress responses, and their potential application in breeding.

Trypsin inhibitor content and activity of soaking water whey as waste in soy milk processing.

Soybean soaking water whey (SWW) is obtained as the waste of soy milk production and mostly represents an environmental problem. The aim of this study was to assess the content of proteins and content and activity of trypsin inhibitors of fresh SWW, obtained during soy milk production. Two zones of Bowman-Birk trypsin inhibitors (BBI) were detected. One was identified as a monomeric form of BBI (0.61-2.93%) and the other one was identified as a polymeric form of BBI (0.45-3.33%). The degree of BBI extraction (1.88-5.49%) was influenced by the soybean genotype and the grain size, i.e. it increased with increasing grain size. Kunitz trypsin inhibitor was not detected. Total proteins were found in traces in SWW (0.03-0.06%). Low residual trypsin inhibitor activity (0.32-0.55%) suggested that SWW can potentially be applied for preparing food or feed. In that case it will not be waste but a cheap functional supplement with BBI as a biologically active component.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Crystallographic structure of a complex between trypsin and a nonapeptide derived from a Bowman-Birk inhibitor found in Vigna unguiculata seeds.

Natural inhibitors of proteases have been classified into different families, among them is the Bowman-Birk Inhibitor (BBI) family. Members of BBI have two structurally reactive loops that simultaneously inhibit trypsin and chymotrypsin. Here, we have investigated the binding of bovine trypsin by a cyclic nonapeptide, named PTRY9 (CTKSIPPQC), derived of the black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) from Vigna unguiculata seeds. This peptide was synthetically produced with the disulfide bond restraining its conformation to mimic the reactive loop that inhibits trypsin. PTRY9 complexed to pancreatic bovine trypsin was crystallized in orthorhombic and trigonal space groups, P212121 and P3221, with maximum resolutions of 1.15 and 1.61 Å, respectively. The structures presented refinement parameters of Rwork = 14.52 % and Rfree = 15.59 %; Rwork = 15.60 % and Rfree = 18.78 %, and different surface area between the peptide and the enzyme of 1024 Å2 and 1070 Å2, respectively. The binding site of the PTRY9 is similar to that found for BTCI as shown by a r.m.s.d. of 0.358 Šbetween the superimposed structures and the electrostatic complementary pattern at the enzyme-peptide interface. Additionally, enzyme inhibition assays show that the affinity of trypsin for PTRY9 is smaller than that for BTCI. In vitro assays revealed that, like BTCI, this synthetic peptide is not cytotoxic for normal mammary epithelial MCF-10A cells, but exerts cytotoxic effects on MDA.MB.231 invasive human breast cancer cells.

In vivo anti-inflammatory efficacy of the combined Bowman-Birk trypsin inhibitor and genistein isoflavone, two biological compounds from soybean.

Soybean Bowman-Birk protease inhibitor (BBI) and genistein, two biological compounds from soybean, are well-known for their anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was designing a BBI-genistein conjugate and then investigating its protective effect on lipopolysaccharide (LPS)-induced inflammation in BALB/c mice, compared with the effects of combination of BBI and genistein. BBI was purified from soybean and the BBI-genistein conjugate was synthesized. The BALB/c mice were intraperitoneally treated 2 hours before LPS induction. Our results showed that treatment with the combination of BBI and genistein greatly led to more reduced serum levels of tumor necrosis factor (TNF)-α and interferon (IFN)-γ compared with the treatments of BBI alone, the BBI-genistein conjugate, and genistein alone, respectively. Moreover, the expression of TNF-α and IFN-γ in the splenocytes was significantly downregulated along with improving host survival against the LPS-induced lethal endotoxemia in the same way. Our data support a new combined therapy using BBI and genistein, as natural anti-inflammatory agents, to develop a new drug for inflammatory diseases.

A Bowman-Birk protease inhibitor purified, cloned, sequenced and characterized from the seeds of Maclura pomifera (Raf.) Schneid.

MAIN CONCLUSION: A new BBI-type protease inhibitor with remarkable structural characteristics was purified, cloned, and sequenced from seeds of Maclura pomifera , a dicotyledonous plant belonging to the Moraceae family. In this work, we report a Bowman-Birk inhibitor (BBI) isolated, purified, cloned, and characterized from Maclura pomifera seeds (MpBBI), the first of this type from a species belonging to Moraceae family. MpBBI was purified to homogeneity by RP-HPLC, total RNA was extracted from seeds of M. pomifera, and the 3'RACE-PCR method was applied to obtain the cDNA, which was cloned and sequenced. Peptide mass fingerprinting (PMF) analysis showed correspondence between the in silico-translated protein and MpBBI, confirming that it corresponds to a new plant protease inhibitor. The obtained cDNA encoded a polypeptide of 65 residues and possesses 10 cysteine residues, with molecular mass of 7379.27, pI 6.10, and extinction molar coefficient of 9105 M-1 cm-1. MpBBI inhibits strongly trypsin with K i in the 10-10 M range and was stable in a wide array of pH and extreme temperatures. MpBBI comparative modeling was applied to gain insight into its 3D structure and highlighted some distinguishing features: (1) two non-identical loops, (2) loop 1 (CEEESRC) is completely different from any known BBI, and (3) the amount of disulphide bonds is also different from any reported BBI from dicot plants.

Oligomerization affects the kinetics and thermodynamics of the interaction of a Bowman-Birk inhibitor with proteases.

The black-eyed pea trypsin/chymotrypsin inhibitor (BTCI) forms concentration dependent homomultimers, as previously demonstrated by Light scattering and Atomic Force Microscopy. Considering that these self-aggregates might influence their binding to cognate enzymes, we investigated the interaction of BTCI at picomolar concentrations using surface immobilized Chymotrypsin (α-CT) and Trypsin (T) by Surface Plasmon Resonance. Our results indicate that BTCI has subnanomolar affinity to both immobilized enzymes, which is approximately two orders of magnitude higher than previously reported. Moreover, we probed the influence of temperature on protein binding equilibria in order to investigate their interaction energetics. While the BTCI/T interaction concurs with the canonical entropy-driven mechanism described for BBI interactions with serine proteinases, the BTCI/α-CT interaction does not. Our measurements indicate that bimolecular BTCI/α-CT complexes form with a negative enthalpy change and a moderate entropic increase. Direct calorimetric evaluation is in accord with the van't Hoff approximation obtained by SPR. We demonstrate that as protein concentrations increase to the micromolar range, secondary endothermic events become prevalent and affect both the kinetics and thermodynamics of protein associations. Our study reinforces that BBI interactions with serine proteinases should be studied in dilute solutions to abridge often neglected secondary interactions.

Oral Cancer Chernoprevention: Current Status and Future Direction.

The aim of this study is to review the current status of cancer chemoprevention and its effectiveness in treatment of oral premalignant lesions and prevention of their progression to oral cancer. The challenges encountered in the different oral cancer chemoprevention clinical trials, including lack of surrogate endpoints, reversal of histologic premalignant changes as study endpoints, tobacco use, human papillomavirus, delivery system, adverse effects and risk of bias in clinical studies, are presented.

Rational tailoring of substrate and inhibitor affinity via ATRP polymer-based protein engineering.

Atom transfer radical polymerization (ATRP)-based protein engineering of chymotrypsin with a cationic polymer was used to tune the substrate specificity and inhibitor binding. Poly(quaternary ammonium) was grown from the surface of the enzyme using ATRP after covalent attachment of a protein reactive, water-soluble ATRP-initiator. This "grafting from" conjugation approach generated a high density of cationic ammonium ions around the biocatalytic core. Modification increased the surface area of the protein over 40-fold, and the density of modification on the protein surface was approximately one chain per 4 nm(2). After modification, bioactivity was increased at low pH relative to the activity of the native enzyme. In addition, the affinity of the enzyme for a peptide substrate was increased over a wide pH range. The massively cationic chymotrypsin, which included up to 2000 additional positive charges per molecule of enzyme, was also more stable at extremes of temperature and pH. Most interestingly, we were able to rationally control the binding of two oppositely charged polypeptide protease inhibitors, aprotinin and the Bowman-Birk trypsin-chymotrypsin inhibitor from Glycine max, to the cationic derivative of chymotrypsin. This study expands upon our efforts to use polymer-based protein engineering to predictably engineer enzyme properties without the need for molecular biology.

Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry.

RATIONALE: The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes. METHODS: Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. RESULTS: Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag). CONCLUSIONS: The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi /yj ion series depending on the positions of the tags.

The glomerular parietal epithelial cell's responses are influenced by SM22 alpha levels.

BACKGROUND: Studies have shown in several diseases initially affecting podocytes, that the neighboring glomerular parietal epithelial cells (PECs) are secondarily involved. The PEC response might be reparative under certain circumstances, yet injurious under others. The factors governing these are not well understood. We have shown that SM22α, an actin-binding protein considered a marker of smooth muscle differentiation, is upregulated in podocytes and PECs in several models of podocyte disease. However, the impact of SM22α levels on PECs is not known. METHODS: Experimental glomerular disease, characterized by primary podocyte injury, was induced in aged-matched SM22α+/+ and SM22α-/-mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody. Immunostaining methods were employed on days 7 and 14 of disease. RESULTS: The number of PEC transition cells, defined as cells co-expressing a PEC protein (PAX2) and podocyte protein (Synaptopodin) was higher in diseased SM22α-/-mice compared with SM22α+/+mice. WT1 staining along Bowman's capsule is higher in diseased SM22α-/-mice. This was accompanied by increased PEC proliferation (measured by ki-67 staining), and an increase in immunostaining for the progenitor marker NCAM, in a subpopulation of PECs in diseased SM22α-/-mice. In addition, immunostaining for vimentin and alpha smooth muscle actin, markers of epithelial-to-mesenchymal transition (EMT), was lower in diseased SM22α-/-mice compared to diseased SM22α+/+mice. CONCLUSION: SM22α levels may impact how PECs respond following a primary podocyte injury in experimental glomerular disease. Absent/lower levels favor an increase in PEC transition cells and PECs expressing a progenitor marker, and a lower EMT rate compared to SM22α+/+mice, where SM22 levels are markedly increased in PECs.

[Production and properties of recombinant glutenin-cleaving proteinases from Eurygaster integriceps Put].

cDNAs coding for a mature form of glutenin-cleaving trypsin-like proteinase (referred to as glutenin-hydrolyzing proteinase 3 or GHP3) from the insect pest-Eurygaster integriceps Put. and a zymogen of this proteinase containing a signal peptide required for protein secretion were cloned into vectors pPIC9 and pPIC3.5, respectively. The constructs were used for protein expression in cells of the methylotrophic yeast Pichia pastoris. The recombinant protein corresponding to the mature form of the proteinase was secreted into the culture medium and possessed proteolytic activity, while the zymogen acquired activity after trypsin, treatment. Both recombinant enzymes hydrolyzed high-molecular weight glutenin subunits from wheat of the variety Ege-88 and a range of other soft and durum wheat varieties. Chymotrypsin inhibitor I from potatoes and related inhibitors from seeds of plants of the subclass Asteridae, the Kunitz-type trypsin inhibitor from soybeans, and bovine aprotinin had a weak inhibitory effect on the recombinant proteinases, while the Bowman-Birk trypsin and chymotrypsin inhibitor from soybeans did not interact with these enzymes:

Evidence that the Bowman-Birk inhibitor from Pisum sativum affects intestinal proteolytic activities in chickens.

Chicken diet essentially relies on soybean as the major source of proteins but there are increasing efforts to identify other protein-rich feedstuffs. Of these, some pea cultivars constitute interesting sources of proteins, although some of them contain antinutritional factors that may compromise the digestibility of their protein content. Consequently, chickens exhibit low performance, while undigested compounds rejected in feces have a negative environmental impact. In this article, we analyzed the intestinal content of chickens fed a pea diet (Pisum sativum) to decipher the mechanisms that could explain such a low digestibility. Using gelatin zymography, we observed that the contents of chicken fed the pea diet exhibit altered proteolytic activities compared with intestinal contents from chickens fed a rapeseed, corn, or soybean diet. This pea-specific profile parallels the presence of a 34 kDa protein band that resists proteolysis during the digestion process. Using mass spectrometry analysis, we demonstrated that this band contains the pea-derived Bowman-Birk protease inhibitor (BBI) and 3 chicken proteases, the well-known chymotrypsinogen 2-like (CTRB2) and trypsin II-P39 (PRSS2), and the yet uncharacterized trypsin I-P38 (PRSS3). All 3 proteases are assumed to be protease targets of BBI. Molecular modeling of the interaction of pea BBI with PRSS2 and PRSS3 trypsins reveals that electrostatic features of PRSS3 may favor the formation of a BBI-PRSS3 complex at physiological pH. We hypothesize that PRSS3 is specifically expressed and secreted in the intestinal lumen to form a complex with BBI, thereby limiting its inhibitory effects on PRSS2 and chymotrypsinogen 2-like proteases. These data clearly demonstrate that in chickens, feedstuff containing active pea BBI affects intestinal proteolytic activities. Further studies on the effects of BBI on the expression of PRSS3 by digestive segments will be useful to better appreciate the impact of pea on intestine physiology and function. From these results, we suggest that PRSS3 protease may represent an interesting biomarker of digestive disorders in chickens, similar to human PRSS3 that has been associated with gut pathologies.

Elemental sulfur concentration can be used as a rapid, reliable, and cost-effective predictor of sulfur amino acid content of soybean seeds.

In this study, we have examined the feasibility of using elemental sulfur content of soybean seeds as a proxy for the overall sulfur amino acid content of soybean seeds. Earlier, we have identified by high throughput ionomic phenotyping several high and low sulfur containing soybean lines from the USDA Soybean Germplasm Collection. Here, we measured the cysteine and methionine content of select soybean lines by high-performance liquid chromatography. Our results demonstrate that those soybean lines which had high elemental sulfur content also had a higher cysteine and methionine content when compared to soybean lines with low elemental sulfur. SDS-PAGE and immunoblot analysis revealed that the accumulation of Bowman Birk protease inhibitor and lunasin in soybean seeds may only be marginally correlated with the elemental sulfur levels. However, we found a positive correlation between the levels of trypsin and chymotrypsin inhibitor activities and elemental sulfur and sulfur amino acid content of the seeds. Thus, elemental sulfur content and/or protease inhibitor activity measurement can be utilized as a rapid and cost-effective method to predict the overall sulfur amino acid content of soybean seeds. Our findings will benefit breeders in their endeavors to develop soybean cultivars with enhanced sulfur amino acid content.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

The contribution of two disulfide bonds in the trypsin binding domain of horsegram (Dolichos biflorus) Bowman-Birk inhibitor to thermal stability and functionality.

The major Bowman-Birk inhibitor (BBIs) of horsegram (Dolichos biflorus) HGI-III, contains seven interweaving disulfides and is extremely stable to high temperatures. The contributions of two disulfide bonds in the trypsin domain to thermal stability and functionality were evaluated using disulfide deletion variants of wild type protein. Thermal denaturation kinetics, differential scanning calorimetry and urea denaturation studies indicate that the absence of either of the two disulfides destabilizes the protein significantly. C20-C66 contributes substantially to both thermal stability and controls trypsin and chymotrypsin inhibitor activity. These two disulfides act in synergy as deletion of both disulfides leads to a complete loss of thermal stability. The data indicate that the two subdomains are not entirely independent of each other. Long range interactions, between the domains are facilitated by C20-C66. The deletion of the disulfide bonds also increased proteolytic susceptibility in a manner similar to the decreased thermal stability. From this study of rHGI a prototype of legume BBIs in can be concluded that among the array of seven evolutionarily conserved disulfide bonds, the disulfide C20-C66 that connects a residue in the trypsin domain with a residue at the border of the same domain plays a dominant role in maintaining functional and structural stability.

Production of horsegram (Dolichos biflorus) Bowman-Birk inhibitor by an intein mediated protein purification system.

The seeds of the legume horsegram (Dolichos biflorus), a protein rich pulse (bean), contain multiple forms of Bowman-Birk inhibitors (protease inhibitors). The major inhibitor HGI-III contains seven interweaving disulfides and is extremely stable to high temperatures. A soluble HGI-III (rHGI) with the native N-terminus was produced using a pTWIN IMPACT™ purification system. Yield of rHGI was improved by introducing a trypsin sepharose affinity chromatography step resulting in ∼670 fold purification. The biochemical characteristics of rHGI point to its close similarity to seed HGI-III not only in its structure but also in its inhibitory characteristics toward bovine trypsin and chymotrypsin. The expression and purification strategy presented here promises to produce BBIs in their natural form for pharmacological and therapeutic use.