scholarly journals Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes

1993 ◽  
Vol 293 (1) ◽  
pp. 187-193 ◽  
Author(s):  
T Kordula ◽  
A Dubin ◽  
H Schooltink ◽  
A Koj ◽  
P C Heinrich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Oligonucleotide Probe ◽  
Protein Sequencing ◽  
Cloning And Expression ◽  
Coding Region ◽  
Elastase Inhibitor ◽  
Escherichia Coli Cells ◽  
Degenerate Oligonucleotide ◽  
Horse Blood

Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into the bacterial expression vector pKK233-2 and expressed in Escherichia coli cells. Transformed bacteria expressed significant amounts of the protein, which was immunoprecipitated with a specific anti-HLEI antiserum. Furthermore, HLEI expressed in bacteria inhibited the activity of elastase but not trypsin.

scholarly journals Molecular Cloning and Expression of Cu/Zn-Containing Superoxide Dismutase from Fasciola hepatica

2000 ◽  
Vol 68 (7) ◽  
pp. 3941-3948 ◽  
Author(s):  
Tong-Soo Kim ◽  
Younghun Jung ◽  
Byoung-Kuk Na ◽  
Ki-Sun Kim ◽  
Pyung-Rim Chung
Keyword(s):  
Superoxide Dismutase ◽  
Amino Acid ◽  
Fasciola Hepatica ◽  
Human Subjects ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Gene Fragment ◽  
Coding Region ◽  
Sod Activity ◽  
Degenerate Oligonucleotide

ABSTRACT The cytosolic superoxide dismutase (SOD) of Fasciola hepatica, a causative agent of fascioliasis, was purified and characterized. The enzyme consists of two identical subunits, each with an apparent molecular mass of 17.5 kDa. An analysis of the enzyme's primary structure and inhibition studies revealed that the enzyme is a copper/zinc-containing SOD (Cu/Zn-SOD). The enzyme activity was relatively stable in a broad pH range, from pH 7.0 to 10.0, and the enzyme showed maximum activity at pH 7.5. This enzyme also displayed strong antigenicity against sera of bovine and human subjects with fascioliasis. The SOD gene fragment was amplified by PCR with degenerate oligonucleotide primers derived from amino acid sequences conserved in the Cu/Zn-SODs of other organisms. An F. hepatica cDNA library was screened with the SOD gene fragment as a probe. As a result, a complete gene encoding the Cu/Zn-SOD was identified, and its nucleotide sequence was determined. The gene had an open reading frame of 438 bp and 146 deduced amino acids. Comparison of the deduced amino acid sequence of the enzyme with previously reported Cu/Zn-SOD amino acid sequences revealed considerably high homologies. The coding region of the F. hepatica Cu/Zn-SOD was cloned and expressed in Escherichia coli. Staining of native polyacrylamide gel for SOD activity of the expressed protein revealed SOD activity that was inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide. This means that the presence of the recombinant fusion protein is indicative of Cu/Zn-SOD. The expressed protein also reacted with sera of bovine and human subjects with fascioliasis, but it did not react with sera of uninfected bovine and human subjects.

scholarly journals Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis)

1986 ◽  
Vol 235 (3) ◽  
pp. 895-898 ◽  
Author(s):  
M S López de Haro ◽  
A Nieto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Untranslated Regions ◽  
Coding Region ◽  
Homologous Proteins ◽  
Lepus Capensis ◽  
Rabbit Gene

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.

Sequence and expression of a stem-abundant caffeic acid O-methyltransferase cDNA from perennial ryegrass (Lolium perenne)

1998 ◽  
Vol 25 (2) ◽  
pp. 225 ◽  
Author(s):  
Fiona M. McAlister ◽  
Colin L. D. Jenkins ◽  
John M. Watson
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Caffeic Acid ◽  
Lolium Perenne ◽  
Perennial Ryegrass ◽  
Leaf Sheath ◽  
Predicted Amino Acid Sequence ◽  
Degenerate Oligonucleotide ◽  
Blot Hybridisation ◽  
Southern Blot Hybridisation

A perennial ryegrass (Lolium perenne) cDNA library was screened with a PCR-amplified comt DNA fragment generated from ryegrass cDNA template using degenerate oligonucleotide primers. A full-length cDNA (LpeComt1) was isolated and shown to encode a caffeic acid O-methyltransferase (COMT) enzyme by analysis of its predicted amino acid sequence. This cDNA was expressed in E.coli and COMT activity was demonstrated in bacterial extracts. The predicted amino acid sequence of the perennial ryegrass COMT was highly similar (88%) with that of a COMT isolated from maize. Southern blot hybridisation analysis using this cDNA indicated that it is a member of a small gene family comprising at least two genes in perennial ryegrass. The LpeComt1 gene is strongly expressed in stem tissue, but not obviously in shoot, leaf blade, leaf sheath, flower or root tissue.

scholarly journals Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 593-600 ◽  
Author(s):  
JP Rosa ◽  
PF Bray ◽  
O Gayet ◽  
GI Johnston ◽  
RG Cook ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Chromosome 17 ◽  
Coding Region ◽  
Membrane Glycoproteins ◽  
Cdna Sequences ◽  
Hel Cells ◽  
Adhesive Proteins

Abstract Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3′ untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.

scholarly journals Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal α-mannosidase B

1995 ◽  
Vol 305 (2) ◽  
pp. 363-366 ◽  
Author(s):  
C Emiliani ◽  
S Martino ◽  
J L Stirling ◽  
B Maras ◽  
A Orlacchio
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rapid Method ◽  
Protein Sequencing ◽  
Subunit Composition ◽  
Partial Sequence ◽  
Sequencing Analysis ◽  
Cloned Cdna

Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.

scholarly journals Occurrence of PG-Lb, a leucine-rich small chondroitin/dermatan sulphate proteoglycan in mammalian epiphyseal cartilage: molecular cloning and sequence analysis of the mouse cDNA

1996 ◽  
Vol 318 (3) ◽  
pp. 909-914 ◽  
Author(s):  
Kazuhiro KURITA ◽  
Tamayuki SHINOMURA ◽  
Minoru UJITA ◽  
Masahiro ZAKO ◽  
Daihei KIDA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Epiphyseal Cartilage ◽  
Newborn Mouse ◽  
Dermatan Sulphate ◽  
Cdna Fragment ◽  
Sulphate Proteoglycan ◽  
C Terminus ◽  
Degenerate Oligonucleotide

PG-Lb is a chondroitin/dermatan sulphate proteoglycan first isolated from chick embryo limb cartilage. It had been assumed that osteoglycin represents its mammalian homologue. However, partial amino acid sequences of a novel proteoglycan from bovine epiphyseal cartilage showed high identity with those of chick PG-Lb (P. Neame, L. Rosenberg and M. Höök, personal communication). Reverse transcriptase PCR using degenerate oligonucleotide primers gave a cDNA fragment that might correspond to mouse PG-Lb. We isolated a clone from a cDNA library of newborn mouse epiphyseal cartilage using the cDNA fragment as a probe. The cloned cDNA was 1430 bp long and contained a 966 bp open reading frame which encoded the core protein consisting of 322 amino acid residues. The deduced amino acid sequence showed a high overall identity with chick PG-Lb (about 62%, reaching about 80% over the carboxyl two-thirds). In addition, the amino acid sequence contained a signal peptide, six cysteine residues at the invariant relative position to chick PG-Lb, six leucine-rich repeats at the carboxyl two-thirds, three possible glycosaminoglycan-attachment sites (two sites at the N-terminal side and one site at the C-terminus) and two possible Asn-glycosylation sites near the C-terminus. Northern-blot analysis demonstrated the specific expression of a 1.5 kb message in cartilage and testis. These structural features and the characteristic expression suggest that the cloned molecule is mouse PG-Lb.

scholarly journals Protein structure and gene cloning of Syncephalastrum racemosum nuclease

1999 ◽  
Vol 339 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Heng-Chien HO ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Direct Sequencing ◽  
Coli Strain ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Computer Search ◽  
Degenerate Primers ◽  
Syncephalastrum Racemosum

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

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