scholarly journals Molecular cloning and expression of a rat hepatic multiple inositol polyphosphate phosphatase

1997 ◽  
Vol 328 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Andrew CRAXTON ◽  
J. James CAFFREY ◽  
William BURKHART ◽  
T. Stephen SAFRANY ◽  
B. Stephen SHEARS
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Catalytic Domain ◽  
Cloning And Expression ◽  
Predicted Amino Acid Sequence ◽  
Rat Tissues ◽  
Inositol Polyphosphate ◽  
Inositol Hexakisphosphate ◽  
Diverse Range ◽  
Amino Acid Region

The characterization of the multiple inositol polyphosphate phosphatase (MIPP) is fundamental to our understanding of how cells control the signalling activities of ‘higher’ inositol polyphosphates. We now describe our isolation of a 2.3 kb cDNA clone of a rat hepatic form of MIPP. The predicted amino acid sequence of MIPP includes an 18 amino acid region that aligned with approximately 60% identity with the catalytic domain of a fungal inositol hexakisphosphate phosphatase (phytase A); the similarity encompassed conservation of the RHGXRXP signature of the histidine acid phosphatase family. A histidine-tagged, truncated form of MIPP was expressed in Escherichia coli and the enzymic specificity of the recombinant protein was characterized: Ins(1,3,4,5,6)P5 was hydrolysed, first to Ins(1,4,5,6)P4 and then to Ins(1,4,5)P3, by consecutive 3- and 6-phosphatase activities. Inositol hexakisphosphate was catabolized without specificity towards a particular phosphate group, but in contrast, MIPP only removed the β-phosphate from the 5-diphosphate group of diphosphoinositol pentakisphosphate. These data, which are consistent with the substrate specificities of native (but not homogeneous) MIPP isolated from rat liver, provide the first demonstration that a single enzyme is responsible for this diverse range of specific catalytic activities. A 2.5 kb transcript of MIPP mRNA was present in all rat tissues that were examined, but was most highly expressed in kidney and liver. The predicted C-terminus of MIPP is comprised of the tetrapeptide SDEL, which is considered a signal for retaining soluble proteins in the lumen of the endoplasmic reticulum; the presence of this sequence provides a molecular explanation for our earlier biochemical demonstration that the endoplasmic reticulum contains substantial MIPP activity [Ali, Craxton and Shears (1993) J. Biol. Chem. 268, 6161-6167].

scholarly journals Bioinformatics Insights Into Microbial Xylanase Protein Sequences

2018 ◽  
Vol 15 (2) ◽  
pp. 275-294
Author(s):  
Deepsikha Anand ◽  
Jeya Nasim ◽  
Sangeeta Yadav ◽  
Dinesh Yadav
Keyword(s):  
Amino Acid ◽  
Phylogenetic Tree ◽  
Catalytic Domain ◽  
Genetic Manipulation ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Molecular Weights ◽  
Aliphatic Index ◽  
Chemical Attributes

Microbial xylanases represents an industrially important group of enzymes associated with hydrolysis of xylan, a major hemicellulosic component of plant cell walls. A total of 122 protein sequences comprising of 58 fungal, 25 bacterial, 19actinomycetes and 20 yeasts xylanaseswere retrieved from NCBI, GenBank databases. These sequences were in-silico characterized for homology,sequence alignment, phylogenetic tree construction, motif assessment and physio-chemical attributes. The amino acid residues ranged from 188 to 362, molecular weights were in the range of 20.3 to 39.7 kDa and pI ranged from 3.93 to 9.69. The aliphatic index revealed comparatively less thermostability and negative GRAVY indicated that xylanasesarehydrophilicirrespective of the source organisms.Several conserved amino acid residues associated with catalytic domain of the enzyme were observed while different microbial sources also revealed few conserved amino acid residues. The comprehensive phylogenetic tree indicatedsevenorganismsspecific,distinct major clusters,designated as A, B, C, D, E, F and G. The MEME based analysis of 10 motifs indicated predominance of motifs specific to GH11 family and one of the motif designated as motif 3 with sequence GTVTSDGGTYDIYTTTRTNAP was found to be present in most of the xylanases irrespective of the sources.Sequence analysis of microbial xylanases provides an opportunity to develop strategies for molecular cloning and expression of xylanase genes and also foridentifying sites for genetic manipulation for developing novel xylanases with desired features as per industrial needs.

scholarly journals Cloning and expression of rat pancreatic β-cell malonyl-CoA decarboxylase

1999 ◽  
Vol 340 (1) ◽  
pp. 213-217 ◽  
Author(s):  
Nicolas VOILLEY ◽  
Raphaël RODUIT ◽  
Raffaela VICARETTI ◽  
Christophe BONNY ◽  
Gérard WAEBER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cloning And Expression ◽  
Rat Tissues ◽  
Distribution Analysis ◽  
Pancreatic Β Cell ◽  
Β Cell ◽  
Methionine Residue ◽  
Malonyl Coa ◽  
Mrna Tissue Distribution ◽  
Wide Range

To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic β-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3ʹ of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic β-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues.

197 MOLECULAR CLONING AND EXPRESSION OF THE CASHMERE GOAT IZUMO GENE

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
R. H. Na ◽  
L. Liang ◽  
L. Fu
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transmembrane Protein ◽  
Northern Blotting ◽  
Cloning And Expression ◽  
Immunoglobulin Superfamily ◽  
Predicted Amino Acid Sequence ◽  
Type I ◽  
Cashmere Goat ◽  
Calculated Molecular Mass

During the fertilization process, complex events are involved in the fusion between the reacted spermatozoa and the mature oocyte. Fusion implies that many proteins are present on the cell membrane of the gametes. Recently, a new protein, Izumo, has been shown to play a role in the sperm–egg fusion. Izumo was identified through the generation of a monoclonal antibody that inhibits this fusion. This protein belongs to the immunoglobulin superfamily type I transmembrane protein. Izumo can be detected only after acrosome reaction at the spermatozoal surface. The cashmere goat Izumo gene was identified and cloned by 3′ and 5′ rapid amplification of cDNA ends-PCR. The expression of cashmere goat Izumo was examined by RT-PCR and Northern blotting. The full-length cDNA of cashmere goat Izumo contains 1536 bp and an open reading frame of 1035 bp, encoding a polypeptide of 344 amino acids with a calculated molecular mass of 38.76 kDa and a theoretical isoelectric point of 8.18. This predicted amino acid sequence showed 89.82% amino acid identity with the bovine Izumo. The deduced amino acid sequence contained 3 conserved domains of the signal peptide, immunoglobulin-like domain, and transmembrane region. Reverse transcription-PCR and Northern blotting analysis showed that cashmere goat Izumo transcripts were highly expressed in the testis, caput, corpus, cauda epididymis. Cashmere goat Izumo may play a role in the biological process of fertilization. This work was supported by the National Natural Science Foundation (No. 30560103 and No.30740043), China, and the China Postdoctoral Science Foundation.

Cloning and expression analysis of Serum Amyloid A (SAA) from Exopalaemon carinicauda (Holthuis, 1950) (Caridea, Palaemonidae)

Crustaceana ◽  
2019 ◽  
Vol 92 (10) ◽  
pp. 1157-1168
Author(s):  
Shu Jie Sun ◽  
Xue Li ◽  
Chun Mei Wang ◽  
Jian Hua Chen ◽  
Huan Gao ◽  
...  
Keyword(s):  
Amino Acid ◽  
Acute Phase ◽  
Serum Amyloid A ◽  
Acute Phase Protein ◽  
Serum Amyloid ◽  
Cloning And Expression ◽  
Predicted Amino Acid Sequence ◽  
Exopalaemon Carinicauda ◽  
Amyloid A ◽  
Phase Protein

Abstract Serum amyloid A (SAA), an acute-phase protein, has been demonstrated to play a critical role in the inflammatory response. However, data regarding its function in crustaceans are still scarce. In the present study, cDNA of SAA from Exopalaemon carinicauda (Holthuis, 1950) (designated as EcSAA) was cloned and characterized. The full-length cDNA was 683 bp, including an open reading frame (ORF) of 267 bp that encoded 88 amino acid residues. The predicted amino acid sequence of EcSAA contained characteristic motifs of the SAA family. The EcSAA was widely expressed in all tissues, with the highest expression in gill and hepatopancreas. In response to the Vibrio anguillarum Bergeman, 1909 challenge, EcSAA mRNA was up-regulated in gill, hepatopancreas, and haemolymph. These results suggest thatEcSAA may be a constitutive and inducible acute-phase protein involved in anti-pathogen responses in E. carinicauda.

Cloning and expression of a broad pH stable GH11 xylanase from Aspergillus niger C71 / Aspergillus niger C71 den geniş pH aralığında dengeli olan GH11 xylanaz klonlaması ve ekspresyonu

2015 ◽  
Vol 40 (4) ◽  
Author(s):  
Guo-Xing Nie ◽  
Jian-Xin Zhang ◽  
Jin-Feng Shan ◽  
Hong Ming ◽  
Dong-Ying Song ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Aspergillus Niger ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Escherichia Coli Bl21

AbstractObjective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-β-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3).Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods. The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0. The amino acid sequences of XYNB were submitted to the ESyPred3D Web Server 1.0 for Homology modeling. The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE.Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa. Phylogenetic analysis of xynB, three-dimensional structure and overlap analysis of XYNB demonstrated that XYNB has a β-jelly-roll architecture, which is more conversation in the catalytic domain of GH11 xylanases, belonging to the family 11 of glycosyl hydrolases. A maximum enzyme activity of 62,242.33 U•mlConclusion: The xynB had been successfully expressed in Escherichia coli BL21, with high enzyme activity and a broad pH stability, and it would be a very good application prospect as the feed enzyme.

Isolation and embryonic expression of an abdominal-A-like gene from the lepidopteran, Manduca sexta

Development ◽  
1991 ◽  
Vol 112 (1) ◽  
pp. 119-129 ◽  
Author(s):  
L.M. Nagy ◽  
R. Booker ◽  
L.M. Riddiford
Keyword(s):  
Amino Acid ◽  
Manduca Sexta ◽  
Abdominal Segment ◽  
Early Embryo ◽  
Predicted Amino Acid Sequence ◽  
Posterior Half ◽  
Larval Stages ◽  
Amino Acid Region ◽  
In Situ Hybridizations

Using sequence homology to the Drosophila Antennapedia gene, we isolated a homeobox-containing gene from the lepidopteran, Manduca sexta. Sequence analysis and in situ hybridizations to tissue sections suggest that the Manduca gene encodes a lepidopteran homologue of the Drosophila Bithorax complex gene abdominal-A. The predicted amino acid sequence of a 76 amino acid region that includes the homeobox and the regions immediately flanking it are identical between the Manduca and Drosophila genes. Northern blots reveal that the manduca abd-A gene is expressed first in the early embryo and continues to be expressed throughout later embryonic and larval stages. In situ hybridizations show that the posterior half of the first abdominal segment marks the anterior border of the Manduca abd-A expression. This expression pattern demonstrates the conservation of parasegments as domains of gene activity in the lepidopteran embryo. The Manduca abd-A expression extends from the posterior half of the first abdominal segment through the tenth abdominal segment, a domain that is greater than that of the Drosophila abd-A expression, and reflects the difference in visible segment number between the two insects.

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