LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

Molecular cloning and expression of genes of Kunitz-type C protease inhibitors from potato

We cloned the products of polymerase chain reaction of the genome DNA of potato (Solanum tuberosum L., Istrinskii cultivar) and isolated 35 clones, which represent copies of eight genes encoding Kunitz type C proteases. Their nucleotide sequences were established. All the genes were found for the first time and designated as PKPI-C1-PKPI-C8. The gene PKPI-C2, which we had earlier presumed to encode the subtilisin PKSI inhibitor, was cloned into pQE30 vector and then expressed in Escherichia coli cells. The recombinant protein PKPI-C2 underwent spontaneous folding and transformation into a soluble state. We purified it to homogeneity by affinity chromatography. The PKPI-C2 protein efficiently inhibited subtilisin Carlsberg activity and did not act on trypsin, chymotrypsin, or papain.     

Chymotrypsin and trypsin inhibitor isolated from potato tubers

Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., cv. Zhukov’s Jubilee) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for subtilisin Carlsberg. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.     

Immunological Comparisons of Chymotrypsin Inhibitor I among Several Genera of the Solanaceae

Microcomplement fixation was employed to compare the immunological differences that occur between purified inhibitor I from potato tubers, the four purified protomers that comprise it, and inhibitor I from tuber and leaf extracts. Total inhibitors of chymotrypsin and trypsin in leaves of seven genera of the Solanaceae were identified by enzymatic assay. In leaves of three genera, Solanum, Lycopersicum, and Datura, chymotrypsin inhibitor I was identified immunologically. In petals of all seven genera inhibitor I was also identified immunologically. With the microcomplement fixation technique inhibitor I from leaf or petal extracts of eight Solanaceae genera were compared. An immunological relationship of inhibitor I among seven of these genera was established.     

Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers

Product of polymerase chain reaction designated as PKPIJ-B was isolated after amplification from genomic DNA of potato (Solarium tuberosum L., Zhukov Jubilee cultivar) using the designed primers. Nucleotide sequence of PKPIJ-B was determined and amino acid sequence of protein was restored. Analysis of this sequence has allowed us to suggest that isolated gene fragment encodes chymotrypsin and trypsin inhibitor protein (PKCI-23 potato Kunitz-type chymotrypsin inhibitor) of potato tubers.     

X-ray Structure Analysis and Characterization of AFUEI,an Elastase Inhibitor from Aspergillus fumigatus

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.     

Introduction and Expression of Cowpea Trypsin Inhibitor (CpTI) Gene in Transgenic Tobacco

A trypsin/chymotrypsin inhibitor (CpTI) was purified from mature cowpea seeds. A full length cDNA clone encoding the trypsin/chymotrypsin inhibitor protein was isolated from a cDNA library that was constructed using poly(A⁺) RNA from developing seeds. Tobacco protoplasts were transformed with a construct composed of CaMV 35S promoter, NPTII gene, CpTI gene, and NOS terminator, using polyethylene glycol (PEG)-mediated direct gene transfer. The CpTI gene has been integrated into the plant genome, and the expressed CpTI protein from transgenic plants was catalytically active.     

Isolation and characterization from potato tubers of two polypeptide inhibitors of serine proteinases

Two polypeptides, isolated to electrophoretic homogeneity from Russet Burbank potato tubers, are powerful inhibitors of pancreatic serine proteinases. One of the inhibitors, called polypeptide trypsin inhibitor, PTI, has a molecular weight of 5100, and inhibits bovine trypsin. The inhibitor is devoid of methionine, histidine, and tryptophan and contains eight half-cystine residues as four disulfide bridges. The second inhibitor, polypeptide chymotrypsin inhibitor II, PCI-II, has a molecular weight of 5700 and powerfully inhibits chymotrypsin. This inhibitor is also devoid of methionine and tryptophan but it contains only six of half-cystines as three disulflde bonds. Both polypeptides strongly inhibit pancreatic elastase. In immunological double diffusion assays, polypeptide trypsin inhibitor and polypeptide chymotrypsin inhibitor II exhibit a high degree of immunological identity (a) with each other, (b) with a polypeptide chymotrypsin inhibitor (PCI-I, M_r 5400) previously isolated from potato tubers, and (c) with inhibitor II, a larger (monomer M_r ~ 12,000) inhibitor of both trypsin and chymotrypsin which has also been previously isolated from potato tubers. The four polypeptide proteinase inhibitors now isolated from Russet Burbank potato tubers cumulatively inhibit all five major intestinal digestive endo- and exoproteinases of animals. The inhibitors are thought to be antinutrients that are present as part of the natural chemical defense mechanisms of potato tubers against attacking pests.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.     

Comparative studies on S-adenosyl-l-methionine binding sites of protein N-methyltransferases,using 8-azido-S-adenosyl-l-methionine as photoaffinity probe

Employing a photoaffinity labeling procedure with 8-azido-S-adenosyl-l-[methyl-³H]methionine (8-N₃-Ado[methyl-³H]Met), the binding sites for S-adenosyl-l-methionine (AdoMet) of three protein N-methyltransferases [AdoMet:myelin basic protein-arginine N-methyltransferase (EC2.1.1.23); AdoMet:histone-arginin N-methyltransferase (EC2.1.1.23); and AdoMet:cytochromec-lysine N-methyltransferase (EC2.1.1.59)] have been investigated. The incorporation of the photoaffinity label into the enzymes upon UV irradiation was highly specific. In order to define the AdoMet binding sites, the photolabeled enzymes were sequentially digested with trypsin, chymotrypsin, and endoproteinase Glu-C. After each proteolytic digestion, radiolabeled peptide from each enzyme was resolved on HPLC first by gradient elution and further purified by an isocratic elution. Retention times of the purified radiolabeled peptides from the three enzymes from the corresponding proteolysis were significantly different, indicating that their sizes and compositions were different. Amino acid composition analysis of these peptides confirmed further that the AdoMet binding sites of these protein N-methyltransferases are quite different.     

Trypsin inhibitor isolated from <Emphasis Type="Italic">Streptomyces misionensis</Emphasis> UMS1 has anti-bacterial activities and activates α-amylase

Protease inhibitors (PI) discovered to be widely distributed in animals, plants and microorganisms, but the information on bacterial PI is scarce. Trypsin inhibitor, named SMTI, was purified from local actinomycete strain (Kuala Lumpur, Malaysia), Streptomyces misionensis UMS1, by acetone precipitation and trypsin-agarose affinity chromatography. Reverse-phase HPLC did not reveal isoinhibitor in purified PI. The purification resulted in a 14 kDa protein, visualized on SDS-PAGE and analyzed with MALDITOF/ TOF. Isoelectric focusing of SMTI demonstrated a single protein with an acidic pI of 6.2. SMTI showed inhibitory activity towards trypsin (74%) and chymotrypsin (41%). Kinetic analysis of trypsin inhibitory activity of SMTI revealed a competitive mechanism with an inhibition constant, Ki of 5 × 10–7 M. SMTI was also shown to have a significant ability to enhance the acitivity of α-amaylase (47%). Furthermore, SMTI exhibited an antibacterial activity against Bacillus cereus, Erwinia, Ralstonia, Salmonella typhi, and Escherichia coli with MIC of 0.06, 0.06, 0.015, 0.12, and 0.48 mg/mL, respectively     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Overexpression of a bacterial chymotrypsin: Application for l-amino acid ester hydrolysis

In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated l-phenylalanine and l-tyrosine methyl ester hydrolysis.     

Hydroxyproline-rich bacterial agglutinin from potato : extraction, purification, and characterization

A protein, extracted from Katahdin potato (Solanum tuberosum L. cv `Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates virulent strains. The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight%) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 ± 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity ([η] = 92.7), the frictional coefficient (f/f_o = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid.     

Purification and antiparasitic activity of a few legume serine proteinase inhibitors: Effect on erythrocyte invasion,schizont rupture and proteolytic processing of the Plasmodium falciparum AMA1 protein

Legume proteinase inhibitors (PI/s) abrogate proteolytic activity of proteins and cause adverse effects on growth. In this study, two legume PIs – Archidendron ellipticum Trypsin Inhibitor (AeTI) and Derris trifoliata Trypsin Chymotrypsin Inhibitor (DtTCI) were purified and used along with standard, Soybean Trypsin Inhibitor (STI), either singly or in combination, as antiplasmodial agents against two Plasmodium forms (Pf 3D7/Pf FCR3). Both AeTI/DtTCI were purified to homogeneity by HPLC and showed over 98% trypsin/chymotrypsin inhibition, respectively. DtTCI severely arrested growth of both the Plasmodium forms (IC50 of 9.59 μM and 16.86 μM, respectively). In addition, combination of DtTCI/AeTI had synergistic effect against both Pf 3D7 (FIC – 0.19) and Pf FCR3 (FIC − 0.23). Combinations of DtTCI/STI and AeTI/STI showed additive effects for the parasite forms. Time-course studies indicated DtTCI and combination of DtTCI/AeTI, to be potent inhibitor of schizont rupture. Antiproteolytic action of the PIs on Pf Apical Merozoite Antigen 1 (AMA1) protein revealed that none of the inhibitors (singly/combinations) affected the primary proteolysis of PfAMA1 protein. Notably however, the normal secondary proteolysis of PfAMA1 was abrogated by both DtTCI (singly/combinations) and AeTI. Incidentally, the proteolytic processing of PfAMA1 remained unaffected following STI treatment.     

Purification and Partial Characterization of Antifungal Peptides from Soybean Endophyte-Paenibacillus sp strain HKA-15

Culture supernatant of soybean nodule endophytic bacterium Paenibacillus sp strain HKA-15 showed the antifungal activity against Rhizoctonia bataticola, the causative agent of charcoal rot disease in soybean. The activity was detected only during the on set of stationary phase (24h post inoculation) in potato dextrose broth. The culture filtrate was extracted with nbutanol, resolved into two compounds by hydrophobic interaction column (Sephadex LH-20) chromatography and purified by reverse phase HPLC. Bioactive fractions collected from preparative HPLC were characterized as cyclic peptide and depsipeptide. No loss of activity was recorded with these metabolites when exposed to proteinase K, glycerol (50%), sodium dodecyl sulphate (1%), triton X-100 (1%) and wide pH range.     

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae

A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernemacarpocapsae. The purified protease had an apparent molecular mass of 30 kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K_m, V_max and k_cat values of 409 μM/min, 0.389 μM/min and 24.9 s⁻¹, respectively. The protease was most active at pH 8.0 and 35 °C, and its proteolytic activity was almost completely reduced after incubation at 75 °C for 30 min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24 h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.     

Identification of granule-bound starch synthase in potato tubers

Starch granules isolated from potato (Solanum tuberosum L.) tubers were extracted with sodium dodecyl sulfate and the extract was analyzed. A major protein with a molecular weight of 60,000 daltons was detected. This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis and specific antibodies were prepared. The anti-60-kilodalton antibodies obtained (a) cross-reacted with the waxy proteins of both maize (Zea mays L.) and grain amaranth (Amaranthus hypochondriacus L.), and (b) inhibited starch synthase activity in partially digested starch granules of the grain amaranth. This evidence strongly suggests that the major 60-kilodalton protein present in potato starch granules represents the granule-bound starch synthase.