The complete amino acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds

Trypsin inhibitor DE-3 from Erythrina latissima seeds contains 172 amino acids, including 4 half-cystine residues, and resembles the Kunitz-type inhibitors. Limited hydrolysis of DE-3 with trypsin at pH 3 produced two fragments, F1 and F2, containing 63 and 109 amino acids, respectively. Amino-terminal sequence studies revealed that F1 was the N-terminal and that F2 was the C-terminal fragment. The complete amino acid sequence of fragments F1 and F2 was then determined on peptides produced by enzymatic digestion with trypsin and Staphylococcus aureus V8 protease. The sequence of trypsin inhibitor DE-3 from E. latissima seeds shows a high degree of homology to those of Kunitz-type trypsin inhibitors from soybeans and winged bean seeds.     

Isolation of a trypsin inhibitor with deletion of N-terminal pentapeptide from the seeds of Momordica cochinchinensis, the Chinese drug mubiezhi.

A trypsin inhibitor, MCCTI-1, with a molecular weight of 3479 Da as determined by mass spectrometry, was isolated from Momordica cochinchinensis seeds with a procedure involving extraction with 5% acetic acid, ammonium sulfate precipitation, ion exchange chromatography on CM-Sepharose and reverse-phase high performance liquid chromatography. The sequence of its first 13 N-terminal amino acid residues was ILKKCRRDSDCPG which was about 85% identical with the sequence of trypsin inhibitor MCTI-1 from Momordica charantia Linn. When compared with the sequences of most other squash family trypsin inhibitors, the sequence of MCCTI-1 was characterized by the deletion of a pentapeptide from the N-terminus. Trypsin inhibitors also existed in seeds of some hitherto uninvestigated Cucurbitaceae species.     

The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.     

The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica)

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.     

The complete amino acid sequence of the major alpha subunit of the lectin from the seeds of Dioclea grandiflora (Mart)

The complete amino acid sequence of the major alpha subunit of the lectin from seeds of Dioclea grandiflora was determined. The sequence was deduced from analysis of peptides derived from the native alpha subunit by digestion with trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and pepsin; and from larger peptides produced by digestion of the citraconylated protein with trypsin. The alpha subunit consists of a single polypeptide chain of 237 amino acids which differs from the sequence of concanavalin in 53 positions. Significant levels of heterogeneity were observed in five positions in the sequence.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

黑豆种子中一种耐热型胰蛋白酶抑制剂的分离及性质表征

应用硫酸铵分级沉降、弱阳交换色谱CM-Sephadex C-50、凝胶过滤 色谱Sephacryl S-200HR、强阳离子高效液相色谱POROS HS-20,从黑豆种 子中分离纯化一种耐热型蛋白酶抑制剂,命名为TSTI.该蛋白的Ｎ末端序列 为DEYSKPCCDLCMCTRRCPPQ,与豆科植物Bowman-Birk型胰蛋白酶抑制剂具有 高度同源性,推测其属于Bowman-Birk型胰蛋白酶抑制剂.SDS－PAGE和IEF 测出TSTI分子量和等电点分别为23.9 kD 和6.2.TSTI对胰蛋白酶有很强的 抑制作用,当二者摩尔比达到1时,胰蛋白酶活力被完全抑制.此外,该蛋 白酶抑制剂具有很强的热稳定性和pH稳定性,在高达100 ℃温度及pH2-12 范围内处理,其活性均不会受到太大影响.TSTI对植物致病菌苹果轮纹病菌 、瓜果腐霉病菌、白菜黑斑病菌、甜瓜枯萎病菌和葡萄灰霉病菌具有抑制 作用.     

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

Purification and characterization of a trypsin inhibitor from seeds of Murraya koenigii

A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 x 10(-9) M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata.     

Cloning and characterization of a novel trypsin inhibitor (BTIomega1) gene from Fagopyrum esculentum.

Based on the amino acid information of trypsin inhibitor of buckwheat (Fagopyrum Esculentum Moench), degenerated primers were designed and a full-length cDNA sequence named BTIomega1 (Buckwheat Trypsin Inhibitor) was amplified from the leaves RNA by using RT-PCR and rapid amplification of cDNA ends (RACE) methods. Sequence analysis shows that the 392 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96 and 93% homology with BWI-1 and BTI-2, a natural trypsin inhibitor from buckwheat seeds. Southern blotting suggested that three copies of BTIomega1 gene existed in the buckwheat genome. Moreover, a predicted secondary structure and 3D-structural model was constructed by homology modeling. To our knowledge, this is the first all-round report of the gene BTIomega1. The novel BTIomega1 gene has been submitted to the GeneBank under Accession No. DQ289792.     

Isolation, characterization, and properties of a trypsin-chymotrypsin inhibitor from amaranth seeds

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaranth—a highly nutritious protein source. The purification of the inhibitor (AmI) was carried out by affinity chromatography on trypsin-Sepharose and by HPLC. AmI is a single-chain protein of 8 kD, as determined by electrophoresis on SDS-polyacrylamide gels and by gel exclusion on Sephadex G-50 column. It is stable at neutral and alkalinepH and is relatively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such asTribolium castaneum andLocusta migratoria, supporting the hypothesis that inhibitors may have evolved as defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partial amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cereal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in comparison to that of other cereal and legume inhibitors suggest that AmI is a member of a new family of serine protease inhibitors. AmI was found to inhibit the anchorage-independent growth of MCF-7 breast cancer cells, suggesting that AmI may have anticarcinogenic activity.     

荞麦胰蛋白酶抑制剂的原核表达及纯化

根据文献报道的荞麦胰蛋白酶抑制剂的氨基酸序列及本研究室先前已获得的部分基因序列设计引物,经过RT-PCR扩增,获得荞麦胰蛋白酶抑制剂编码区基因全序列.将该基因克隆到原核表达载体pQE-31中,并转化至大肠杆菌M15,经IPTG诱导表达获得可溶性目的蛋白,其表达量约占菌体总蛋白的25%.该目的蛋白经Ni2 -NTA柱亲和纯化,SDS-PAGE分析显示,在大约9kD处出现明显的目的条带,与预计蛋白分子量大小一致.Western blot鉴定证实,目的蛋白N端带有6个组氨酸标签.活性测定表明,目的蛋白具有专一性的胰蛋白酶抑制剂活性,抑制活性约为77U/mg纯化蛋白.本实验为进一步研究荞麦胰蛋白酶抑制剂结构与功能的关系奠定了基础.     

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.     

The amino acid sequence of a Bowman-Birk type proteinase inhibitor from faba beans (Vicia faba L.).

The amino acid sequence of a Bowman-Birk type proteinase inhibitor (FBI) from seeds of faba bean (Vicia faba L.) was determined by analysis of peptide fragments generated by reduction and S-carboxymethylation of enzymatically modified inhibitors, which were obtained from native FBI by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin at pH 3.5. The established sequence showed that FBI is highly homologous with Vicia angustifolia inhibitor (VAI0 but lacks the portion corresponding to the C-terminal 9 amino acids of VAI. The trypsin reactive-site peptide bond in FBI was also indicated to be Lys(16)-Ser(17) and the chymotrypsin reactive-site peptide bond to be Tyr(42)-Ser(43) by limited proteolysis with TPCK-trypsin or TLCK-chymotrypsin and by sequence comparison with other Bowman-Birk type inhibitors.     

The complete amino acid sequence of ribonuclease from the seeds of bitter gourd (Momordica charantia)

The complete amino acid sequence of ribonuclease (RNase MC) from the seeds of bitter gourd (Momordica charantia) has been determined. This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with trypsin, lysylendopeptidase, and chymotrypsin, as well as by chemical cleavage with cyanogen bromide. The protein contains 191 amino acid residues and has a calculated molecular mass of 21,259 Da. Comparison of this sequence with sequences of the fungal RNases, RNase T2, and RNase Rh, revealed that there are highly conserved residues at positions 32-38 (TXHGLWP) and 81-92 (FWXHEWXKHGTC). Furthermore, the sequence of RNase MC was found to be homologous to those of Nicotiana alata S-glycoproteins involved in self-incompatibility sharing 41% identical residues.     

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Production of recombinant human pancreatic secretory trypsin inhibitor by Escherichia coli

A synthetic gene for human pancreatic secretory trypsin inhibitor (PSTI) was fused to the coding sequence for the amino-terminal 135 amino acid residues of human interferon-gamma (IFN-gamma) by interposing a methionine codon sequence, and the resulting hybrid gene was efficiently expressed in Escherichia coli cells. Recombinant human PSTI (rHu-PSTI) was separated from the IFN-gamma/PSTI fused protein by cleavage at the methionine residue with cyanogen bromide. Finally, rHu-PSTI was purified by affinity chromatography on a bovine trypsin-CH-Sepharose 4B column. The amino acid composition, partial amino-terminal sequence, disulfide formation, human trypsin inhibitory activity, and immunoreactivity against rabbit anti-human PSTI serum of rHu-PSTI corresponded to those of the natural form.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.