Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3mg purified (His)(6)-tagged CI-b1 from 200ml of culture medium. Studies on (His)(6)-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against alpha-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.     

Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus

Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions.     

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.     

Jasmonic acid-inducible gene expression of a Kunitz-type proteinase inhibitor in potato tuber disks

Messenger RNAs of a potato (Solanum tuberosum L.) Kunitz-type proteinase inhibitor(s) (PKPI) were present in potato disks excised from tubers stored for 14 months (old tubers) or 2 months (young tubers) after harvest, and disappeared during the aseptic culture. The PKPI mRNA accumulation was found to be induced in potato disks from the old tubers by the addition of jasmonic acid (JA) [3-oxo-2-(2-cis-pentenyl)-cyclopentane-1-acetic acid].     

Purification and characterization of a novel chymotrypsin inhibitor controlled by the chymotrypsin inhibitor A (Ict-A) gene from the larval hemolymph of the silkworm,Bombyx mori

• 1.1. On polyacrylamide gel electrophoresis, chymotrypsin inhibitors in the larval hemolymph of the silkworm were found as 15 electrophoretically distinct bands.
• 2.2. One of them, CI-13 migrating fastest toward the anode, was purified from the hemolymph.
• 3.3. The inhibitor was a monomeric protein with a molecular mass of 14,000 and showed stability for both heat and a wide range of pH. The isoelectric point was pH 4.1.
• 4.4. CI-13 was able to inhibit chymotrypsin completely and trypsin slightly, but was ineffective against other proteinases such as papain, ficin, carboxypeptidase A, V8 proteinase, serratia peptidase, cocoonase and proteinases derived from gut juice of the silkworm.

     

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 Å resolution were obtained using hanging drop method by vapor diffusion at 18 °C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function.     

Genomic structure and expression analysis of the spastic paraplegia gene, SPG7

SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages. Electronic Publication     

Genomic structure and chromosome location of the gene encoding mouse CD59

The gene encoding the mouse analogue of the human complement regulator CD59 was cloned using a combination of long range PCR and genomic library screening. Sequence obtained showed that its genomic structure closely resembled that of the human CD59 gene, comprising 4 exons, each separated by a long intron region. The sizes of introns and exons were comparable to those of the human gene with the exception of the third intron which is 2.5 kb in the mouse compared to 7 kb in the human gene. All exon/intron boundaries conformed to the GT-AG rules for splicing. Radiation hybrid mapping localised mouse Cd59 between D2Mit333 and D2Mit127 on chromosome 2, a region homologous with human chromosome 11p13 where the human CD59 gene is localised. These data have permitted the construction of a gene targeting vector for the generation of transgenic mice deficient in CD59.     

Amino-acid sequences of two basic chymotrypsin inhibitors from silkworm larval hemolymph

Amino-acid sequences of two basic chymotrypsin inhibitors from silkworm hemolymph (SCI-I and SCI-II) are determined. They are composed of each 62 amino-acid residues with differences in only two positions to each other. They both contain six half cystines in a similar arrangement as that of Kunitz-type proteinase inhibitor, except for the one amino-acid insertion in the first cysteine frame. The inhibitory activity of SCI-II against trypsin should be attributed to Lys44 displacing Gln44 in SCI-I which has no antitryptic activity.     

Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.     

Taiwan cobra chymotrypsin inhibitor: cloning, functional expression and gene organization

A cDNA encoding chymotrypsin inhibitor was constructed from the cellular RNA isolated from the venom glands of Naja atra (Taiwan cobra). The resultant amino acid sequence showed that the mature protein is comprised of 57 amino acid residues with six cysteine residues. Cloned protein was expressed and isolated from the inclusion bodies of E. coli and refolded into a functional protein in vitro. Deleting the first three residues at its N-terminus caused a moderate increase in the inhibitory constant (K(i)) against chymotrypsin. The genomic DNA encoding the chymotrypsin inhibitor was amplified by PCR. The gene shares virtually an identical structural organization with the beta-bungarotoxin B1 chain (a snake Kunitz/BPTI neurotoxic homolog) gene. Moreover, the overall sequence identity of the N. atra chymotrypsin inhibitor and beta-bungarotoxin B1 chain genes was up to 83%. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Primary structure and antiproteolytic activity of a Kunitz-type inhibitor from bovine spleen

The amino acid sequence of protease inhibitor II, previously isolated from bovine spleen, has been completely elucidated and reveals a high homology (approximately 90%) with that of bovine pancreatic trypsin inhibitor (BPTI), the well-known Kunitz inhibitor. The secondary and tertiary structure of this new inhibitor appears similar to that of BPTI. Whereas its affinity for bovine trypsin, chymotrypsin, and trypsinogen is almost identical to that of BPTI, the affinity for porcine pancreatic kallikrein is decreased, as expected on the basis of the amino acid substitutions. Analysis of the pH dependence of the affinity constant confirms the previous assignment of the ionizable groups, whose pK values are perturbed on complex formation, to kallikrein and not to the inhibitor molecule.     

Genomic structure, chromosome mapping and expression analysis of the human AXIN2 gene

Conductin is a Wnt signalling protein and serves as a negative regulator of beta-catenin stability. We have previously isolated the human homolog (AXIN2) of the murine conductin gene and shown that it is mutated in colorectal cancer (CRC) with defective mismatch repair (MMR). Here we report the detailed genomic structure of this gene by analysis of cDNA and genomic clones. The gene spans > or =25 kb containing ten exons ranging from 96 bp to 904 bp. All splice donor and acceptor sites conform to the GT/AG rule. FISH (Fluorescence in situ Hybridization) analysis localized this gene to human chromosome band 17q24 and showed that it exists as a single copy in the human genome. Northern blot analysis from different human organs demonstrated that the AXIN2 gene is highly expressed in human thymus, prostate, testis, small intestine and ovarian tissues but expressed at a lower level in colon. The data reported here provides a framework for further analysis of this important Wnt signalling protein in vertebrate development and tumorigenesis.     

Genomic organization, expression analysis, and chromosomal localization of the mouse PEX3 gene encoding a peroxisomal assembly protein

The peroxin Pex3p has been identified as an integral peroxisomal membrane protein in yeast where pex3 mutants lack peroxisomal remnant structures. Although not proven in higher organisms, a role of this gene in the early peroxisome biogenesis is suggested. We report here the cDNA cloning and the genomic structure of the mouse PEX3 gene. The 2 kb cDNA encodes a polypeptide of 372 amino acids (42 kDa). The gene spans a region of 30 kb, contains 12 exons and 11 introns and is located on band A of chromosome 10. The putative promoter region exhibits characteristic housekeeping features. PEX3 expression was identified in all tissues analyzed, with the strongest signals in liver and in testis, and could not be induced by fenofibrate. The data presented may be useful for the generation of a mouse model defective in PEX3 in order to clarify the yet unknown functional impact of disturbances in early peroxisomal membrane assembly.