Molecular engineering of a small trypsin inhibitor based on the binding loop of horsegram seed Bowman-Birk inhibitor.

CONTEXT: The Bowman-Birk inhibitors (BBIs) are currently investigated with renewed interest due to their therapeutic properties in cancer and other inflammatory disease treatment. The molecular mass of the BBI is a limitation, as sufficient amounts of the inhibitor do not reach the organs outside the gastrointestinal tract when administered orally. METHOD: The anti-tryptic domain of HGI-III of horsegram (Dolichos biflorus) was cloned using the vector pET-20b (+) and expressed in E. coli BL21 (DE3) pLysS. RESULTS: Kinetic analysis of this anti-tryptic peptide (recombinant trypsin inhibitory domain (rTID)) reveals that it is a potent inhibitor of trypsin and human tryptase. The K(i) (3.2 ± 0.17 × 10(-8) M) establishes a very high affinity to bovine trypsin. rTID inhibited human lung tryptase (IC(50) 3.78 ± 0.23 × 10(-7) M). The rTID is resistant to the digestive enzymes found in humans and animals. CONCLUSION: These properties propagate further research on the use of rTID as a therapeutic for cancer and other related inflammatory diseases.

Generation and identification of variants with improved purification yield of Bowman-Birk protease inhibitors carrying protein binding loops.

Replacing the chymotrypsin inhibitory loop of soybean Bowman-Birk inhibitor (sBBI) with a VEGF binding peptide (BBI-AV) significantly reduces the overall purification yield when BBI-AV is produced as a fusion protein in a Bacillussubtilis expression system. The low purification yield is primarily due to a higher fraction of molecules with incorrect disulfide bond configurations after production and also after disulfide bond shuffling induced by 2-mercaptoethanol. To improve production yields, site-saturation libraries were generated at 39 out of the 66 amino acid residues of BBI-AV. Initial screens were designed to select for variants with higher trypsin inhibitory activities than the parent after treatment with a reducing agent. Secondary screens were developed to select for variants with the highest purification yields, and to also eliminate any false positives. From the screens, it was found that positively charged substitutions in the exposed hydrophobic patch region (sites 27, 29, 40, 50 & 52) are especially productive. In fact, one substitution, F50R, improves the purification yield to nearly the same level as wild-type sBBI. Productive amino acid substitutions were combined to select for the variant with the best overall yield after purification. Several variants were obtained with higher purification yields than even sBBI. The octuple variants, A13I-S25R-M27A-L29P-S31A-A40H-F50K-V52T and A13I-S25K-M27A-L29R-S31E-A40K-F50Q-V52Q, are particularly productive having greater than a five fold increase in final purification yield over the parent.

A Bacillus subtilis fusion protein system to produce soybean Bowman-Birk protease inhibitor.

A fusion protein based expression system was developed in the Gram-positive bacterium Bacillus subtilis to produce the soybean Bowman-Birk protease inhibitor (sBBI). The N-terminus of the mature sBBI was fused to the C-terminus of the 1st cellulose binding domain linker (CBD linker) of the BCE103 cellulase (from an alkalophilic Bacillus sp.). The strong aprE promoter was used to drive the transcription of the fusion gene and the AprE signal sequence was fused to the mature BCE103 cellulase for efficient secretion of the fusion protein into the culture medium. It was necessary to use a B. subtilis strain deficient in nine protease genes in order to reduce the proteolytic degradation of the fusion protein during growth. The fusion protein was produced in shake flasks at concentrations >1g/L. After growth, the sBBI was activated by treatment with 2-mercaptoethanol to allow the disulfide bonds to form correctly. An economical and scalable purification process was developed to purify sBBI based on acid precipitation of the fusion protein followed by acid/heat cleavage of the fusion protein at labile Asp-Pro bonds in the CBD linker. If necessary, non-native amino acids at the N- and C-termini were trimmed off using glutamyl endopeptidase I. After purification, an average of 72 mg of active sBBI were obtained from 1L of culture broth representing an overall yield of 21% based on the amount of sBBI activated before purification.

Tobacco ribosomal DNA spacer element elevates Bowman-Birk inhibitor expression in tomato plants.

Amplification promoting sequence (aps), from tobacco rDNA, was found to induce amplification and enhances the expression of heterologous genes, consequently increasing the expression of transgenic proteins in tobacco. In this report we demonstrate that aps element also affects integration, transcription, and translation of a soybean protease inhibitor, Bowman-Birk inhibitor (BBI), in transgenic tomato plants and quantifies its effects in different expression vectors. A synthetic bbi gene was constructed, based on the wild-type gene containing two independent inhibition sites; trypsin and chymotrypsin. Transformation vectors were designed using two different promoters; the tomato fruit specific E8 promoter and the constitutively active 35S CaMV promoter. These vectors were transformed into 'Moneymaker' tomato plants. In tomato fruits and leaves, aps caused a 3-fold increase in bbi mRNA levels when compared to the lines without aps. Similar increases were obtained in plants expressing bbi controlled by E8 or 35S CaMV promoters. Also, the level of BBI protein expression in aps-transformed plants was 3 fold-higher than in plants without aps. This is the first report of aps effect on the enhanced gene expression and transgenic protein production in plant other than tobacco.

Role of jasmonate in the rice (Oryza sativa L.) self-defense mechanism using proteome analysis.

Exogenously applied jasmonic acid (JA) was used to study changes in protein patterns in rice (Oryza sativa L.) seedling tissues, to classify these changes, and to assign a role for these changes, in order to define the role of JA in the rice self-defense mechanism. High resolution two-dimensional polyacrylamide gel electrophoretic analysis revealed induction of new proteins in both leaf and stem tissues after JA treatment, with the major protein spots further analyzed through N-terminal and internal amino acid sequencing, purification, antibody production, and immunoblot analysis. JA treatment results in necrosis in these tissues, which is accompanied by drastic reductions in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) subunits, and was confirmed using immunoblotting. Induction of novel proteins was found particularly in the stem tissues, including a new basic 28 kDa Bowman-Birk proteinase inhibitor protein (BBPIN; jasmonate-induced stem protein, JISP 6), and acidic 17 kDa pathogenesis-related class 1 protein (PR-1, JISP 9). This induction of proteins was blocked by a protein synthesis inhibitor cycloheximide (CHX) indicating de novo protein synthesis. Kinetin (KIN), a cytokinin and free radical scavenger reversed RuBisCO decreases, but not induction of proteins. Immunoblot analysis using antibodies generated against these purified proteins revealed a tissue-specific expression pattern and time-dependent induction after JA treatment. Our results indicate that jasmonate affects defense-related gene expression in rice seedlings, as evidenced by de novo synthesis of novel proteins with potential roles in plant defense.