scholarly journals Molecular cloning and expression of a unique rabbit osteoclastic phosphotyrosyl phosphatase

1996 ◽  
Vol 316 (2) ◽  
pp. 515-523 ◽  
Author(s):  
Li-Wha WU ◽  
David J. BAYLINK ◽  
K.-H. William LAU
Keyword(s):  
Fusion Protein ◽  
Catalytic Domain ◽  
Osteoclast Formation ◽  
Full Length ◽  
Cloning And Expression ◽  
Regulatory Role ◽  
Rabbit Brain ◽  
Extracellular Region ◽  
Tyrosyl Phosphorylation

Tyrosyl phosphorylation plays an important regulatory role in osteoclast formation and activity. Phosphotyrosyl phosphatases (PTPs), in addition to tyrosyl kinases, are key determinants of intracellular tyrosyl phosphorylation levels. To identify the PTP that might play an important regulatory role in osteoclasts, we sought to clone an osteoclast-specific PTP. A putative full-length clone encoding a unique PTP (referred to as PTP-oc) was isolated from a 10-day-old rabbit osteoclastic cDNA library and sequenced. A single open reading frame predicts a protein with 405 amino acid residues containing a putative extracellular domain, a single transmembrane region, and an intracellular portion. PTP-oc is structurally unique in that, unlike most known transmembrane PTPs, it has a short extracellular region (eight residues), lacks a signal peptide proximal to the N-terminus, and contains only a single ‘PTP catalytic domain’. The PTP catalytic domain shows 45–50% sequence identity with the catalytic domain of human HPTPβ and with the first catalytic domain of LCA. The PTP-oc gene exists as a single copy in the rabbit genome. The corresponding mRNA (3.8 kb) is expressed in osteoclasts but not in other bone-derived cells (e.g. osteoblasts and stromal cells). The 3.8 kb PTP-oc mRNA transcript was also expressed in the rabbit brain, kidney and spleen. However, the brain and kidney, but not osteoclasts or spleen, also expressed a larger transcript (6.5 kb). The PTP catalytic domain of PTP-oc was expressed as a GST–cPTP-oc fusion protein. In vitro phosphatase assays indicated that the purified fusion protein exhibited phosphatase activities at neutral pH values toward p-nitrophenyl phosphate, phosphotyrosyl Raytide, and phosphotyrosyl histone, whereas it had no appreciable activity toward phosphoseryl casein. In summary, we have: (a) cloned and sequenced the putative full-length cDNA of a unique PTP (PTP-oc) from rabbit osteoclasts; (b) shown that the mature 3.8 kb PTP-oc mRNA was expressed primarily in osteoclasts and the spleen; and (c) shown that the PTP-oc fusion protein exhibited a phosphotyrosine-specific phosphatase activity. In conclusion, PTP-oc represents a structurally unique subfamily of transmembrane PTPs.

scholarly journals Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus

1996 ◽  
Vol 319 (3) ◽  
pp. 713-716 ◽  
Author(s):  
Stephen THOMAS ◽  
Salvador SORIANO ◽  
Clive d'SANTOS ◽  
George BANTING
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Catalytic Domain ◽  
Maltose Binding Protein ◽  
Full Length ◽  
Regulatory Role ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Inositol 1,4,5 Trisphosphate ◽  
N Terminus

We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli. The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 126-126 ◽  
Author(s):  
Weirui Zhang ◽  
David Motto ◽  
David Ginsburg
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Expression Vector ◽  
Full Length ◽  
Mammalian Expression ◽  
Partial Denaturation

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

scholarly journals Purification and characterization of avian glycolipid: beta-galactosyltransferases (GalT-4 and GalT-3): cloning and expression of truncated betaGalT-4.

1998 ◽  
Vol 45 (2) ◽  
pp. 451-467 ◽  
Author(s):  
S S Basu ◽  
S Dastgheib ◽  
S Ghosh ◽  
M Basu ◽  
P Kelly ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Fusion Protein ◽  
Catalytic Domain ◽  
Peptide Mapping ◽  
Cloning And Expression ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Developmentally Regulated ◽  
Peptide Backbone ◽  
Protein Fingerprinting

Acidic glycolipid of ganglio-(containing sialic acid) and sialyl-lactofucosyl-type, SA-Lex (containing sialic acid and fucose) are developmentally regulated and appear to be ubiquitous on neuronal and cancer cell surfaces of animals. Two glycolipid: beta-galactosyltransferases, GalT-3 and GalT-4, were characterized in embryonic chicken brain (ECB). Based on substrate competition experiments, these two activities were believed to be due to expression of two gene products. A cDNA fragments (about 600 bp) encoding the catalytic domain of the GalT-4 (UDP-Gal:LcOse3Cer beta1,4galactosyltransferase) from ECB and human Colo-204 were isolated. These cDNAs were expressed as a soluble glutathione-S-transferase fusion protein (48 kDa) in Escherichia coli. Interactions between GlcNAc-, UDP-hexanolamine-, and alpha-lactalbumin were studied with the purified fusion protein (recombinant and truncated). Functionally it was similar to that of native GalT-4 purified (40000-fold) from 11-day-old ECB. GalT-3 (UDP-Gal:GM2beta1,3galactosyltransferase) was purified from 19-day-old ECB, and a polyclonal antibody was produced against the peptide backbone for immunoscreening of a lambdaZAP ECB cDNA expression library. Each of the GalT-3 peptides (62 and 65 kDa was analyzed by protein fingerprinting analysis indicating a similar peptide mapping pattern.

scholarly journals Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms

2019 ◽  
Vol 34 (5) ◽  
pp. 482-496 ◽  
Author(s):  
Gaili Guo ◽  
Kankan Wang ◽  
Shan-Shan Hu ◽  
Tian Tian ◽  
Peng Liu ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Biochemical Properties ◽  
Period Change ◽  
Casein Kinase ◽  
Full Length ◽  
Casein Kinase 1 ◽  
Autokinase Activity

Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.

scholarly journals The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

2003 ◽  
Vol 185 (15) ◽  
pp. 4418-4423 ◽  
Author(s):  
Anne Marie Di Guilmi ◽  
Andréa Dessen ◽  
Otto Dideberg ◽  
Thierry Vernet
Keyword(s):  
Streptococcus Pneumoniae ◽  
Pathogenic Bacteria ◽  
Catalytic Domain ◽  
Soluble Form ◽  
Lipid Ii ◽  
Extracellular Region ◽  
Catalytically Active ◽  
Bacterial Peptidoglycan

ABSTRACT The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N-acetylglucosaminyl-N-acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat/K m were measured at 40.6 μM (± 15.5) and 1 × 10−3 M−1 s−1, respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S. pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

scholarly journals Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudo-modularity

2021 ◽  
Author(s):  
Petko M. Petkov ◽  
Natalie R. Powers ◽  
Timothy Billings ◽  
Kenneth Paigen
Keyword(s):  
Tertiary Structure ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Female Fertility ◽  
Full Length ◽  
Set Domain ◽  
Methyltransferase Activity ◽  
The Difference

PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, glu360pro (Prdm9EP) and glu360lys (Prdm9EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9EP is slightly less hypomorphic than Prdm9EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9EP, but not Prdm9EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9EP and Prdm9EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain versus the full-length protein show that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

scholarly journals Purification and Functional Characterization of a Biologically Active Full-Length Feline Immunodeficiency Virus (FIV) Pr50Gag

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 689 ◽  
Author(s):  
Anjana Krishnan ◽  
Vineeta N. Pillai ◽  
Akhil Chameettachal ◽  
Lizna Mohamed Ali ◽  
Fathima Nuzra Nagoor Pitchai ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Protein Interactions ◽  
Feline Immunodeficiency Virus ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Full Length ◽  
Biologically Active ◽  
Genomic Rna ◽  
Immunodeficiency Virus

The feline immunodeficiency virus (FIV) full-length Pr50Gag precursor is a key player in the assembly of new viral particles. It is also a critical component of the efficient selection and packaging of two copies of genomic RNA (gRNA) into the newly formed virus particles from a wide pool of cellular and spliced viral RNA. To understand the molecular mechanisms involved during FIV gRNA packaging, we expressed the His6-tagged and untagged recombinant FIV Pr50Gag protein both in eukaryotic and prokaryotic cells. The recombinant Pr50Gag-His6-tag fusion protein was purified from soluble fractions of prokaryotic cultures using immobilized metal affinity chromatography (IMAC). This purified protein was able to assemble in vitro into virus-like particles (VLPs), indicating that it preserved its ability to oligomerize/multimerize. Furthermore, VLPs formed in eukaryotic cells by the FIV full-length Pr50Gag both in the presence and absence of His6-tag could package FIV sub-genomic RNA to similar levels, suggesting that the biological activity of the recombinant full-length Pr50Gag fusion protein was retained in the presence of His6-tag at the carboxy terminus. Successful expression and purification of a biologically active, recombinant full-length Pr50Gag-His6-tag fusion protein will allow study of the intricate RNA-protein interactions involved during FIV gRNA encapsidation.

scholarly journals Differential effects of two catalytic mutations on full-length PRDM9 and its isolated PR/SET domain reveal a case of pseudo-modularity

Genetics ◽  
2021 ◽  
Author(s):  
Natalie R Powers ◽  
Timothy Billings ◽  
Kenneth Paigen ◽  
Petko M Petkov
Keyword(s):  
Tertiary Structure ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Female Fertility ◽  
Full Length ◽  
Set Domain ◽  
Methyltransferase Activity ◽  
The Difference

Abstract PRDM9 is a DNA-binding histone methyltransferase that designates and activates recombination hotspots in mammals by locally trimethylating lysines 4 and 36 of histone H3. In mice, we recently reported two independently produced point mutations at the same residue, glu360pro (Prdm9 EP) and glu360lys (Prdm9 EK), which severely reduce its H3K4 and H3K36 methyltransferase activities in vivo. Prdm9 EP is slightly less hypomorphic than Prdm9 EK, but both mutations reduce both the number and amplitude of PRDM9-dependent H3K4me3 and H3K36me3 peaks in spermatocytes. While both mutations cause infertility with complete meiotic arrest in males, Prdm9 EP, but not Prdm9 EK, is compatible with some female fertility. When we tested the effects of these mutations in vitro, both Prdm9 EP and Prdm9 EK abolished H3K4 and H3K36 methyltransferase activity in full-length PRDM9. However, in the isolated PRDM9 PR/SET domain, these mutations selectively compromised H3K36 methyltransferase activity, while leaving H3K4 methyltransferase activity intact. The difference in these effects on the PR/SET domain versus the full-length protein show that PRDM9 is not an intrinsically modular enzyme; its catalytic domain is influenced by its tertiary structure and possibly by its interactions with DNA and other proteins in vivo. These two informative mutations illuminate the enzymatic chemistry of PRDM9, and potentially of PR/SET domains in general, reveal the minimal threshold of PRDM9-dependent catalytic activity for female fertility, and potentially have some practical utility for genetic mapping and genomics.

scholarly journals Molecular cloning and expression of a human hST8Sia VI (α2,8-sialyltransferase) responsible for the synthesis of the diSia motif on O-glycosylproteins

2005 ◽  
Vol 392 (3) ◽  
pp. 665-674 ◽  
Author(s):  
Mélanie Teintenier-Lelièvre ◽  
Sylvain Julien ◽  
Sylvie Juliant ◽  
Yann Guerardel ◽  
Martine Duonor-Cérutti ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cell Line ◽  
Cancer Cell Line ◽  
Full Length ◽  
Cloning And Expression ◽  
Soluble Form ◽  
Amino Acid Residues ◽  
Acetylneuraminic Acid ◽  
Mammary Cell

Based on BLAST analysis of the human and mouse genome databases using the human CMP sialic acid; α2,8-sialyltransferase cDNA (hST8Sia I; EC 2.4.99.8), a putative sialyltransferase gene, was identified on human chromosome 10. The genomic organization was found to be similar to that of hST8Sia I and hST8Sia V. Transcriptional expression analysis showed that the newly identified gene was constitutively expressed at low levels in various human tissues and cell lines. We have isolated a full-length cDNA clone from the breast cancer cell line MCF-7 that encoded a type II membrane protein of 398 amino acid residues with the conserved motifs of sialyltransferases. We have established a mammary cell line (MDA-MB-231) stably transfected with the full-length hST8Sia VI and the analysis of sialylated carbohydrate structures expressed at the cell surface clearly indicated the disappearance of Neu5Acα2-3-sialylated structures. The transient expression of a truncated soluble form of the enzyme in either COS-7 cells or insect Sf-9 cells led to the production of an active enzyme in which substrate specificity was determined. Detailed substrate specificity analysis of the hST8Sia VI recombinant enzyme in vitro, revealed that this enzyme required the trisaccharide Neu5Acα2-3Galβ1-3GalNAc (where Neu5Ac is N-acetylneuraminic acid and GalNAc is N-acetylgalactosamine) to generate diSia (disialic acid) motifs specifically on O-glycans.

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