scholarly journals Cloning, Expression, and Immunological Characterization of the P30 Protein of Mycoplasma pneumoniae

2007 ◽  
Vol 15 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Avanish Kumar Varshney ◽  
Rama Chaudhry ◽  
Sushil Kumar Kabra ◽  
Pawan Malhotra
Keyword(s):  
Fusion Protein ◽  
Mycoplasma Pneumoniae ◽  
Transmembrane Protein ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Asthmatic Patients ◽  
C Terminus

ABSTRACT Mycoplasma pneumoniae, a self-replicating cell wall-deficient prokaryote, has a differentiated terminal organelle that is essential for cytadherence and gliding motility. P30, an important protein associated with the terminal organelle, is required for the cytadherence and virulence of M. pneumoniae. P30 is a transmembrane protein with an intracytoplasmic N terminus and an exposed C terminus. In the present study, we amplified and sequenced the full-length p30 gene of Mycoplasma pneumoniae directly from 18 Indian asthmatic patients. Sequence diversity was observed in the p30 genes from 16 clinical samples when the sequences were compared with the sequence of strain M-129. We also successfully expressed a fragment of the p30 gene (P30B) that includes the complete C-terminal proline-rich amino acid sequences in different Escherichia coli expression systems. The maltose binding protein (MBP)-P30B fusion protein was recognized by M. pneumoniae-infected patient sera in immunoblots, and the protein was immunogenic in mice. We further analyzed the reactivity of the MBP-P30B fusion protein with patient sera in an enzyme-linked immunosorbent assay (ELISA) and compared it with the reactivity obtained with a commercial kit (the Serion ELISA Classic kit). The sensitivity and the specificity of the in-house ELISA were 78.57% and 89.47%, respectively. This study suggests that the P30 protein can be used as an antigen along with other adhesin proteins for the immunodiagnosis of M. pneumoniae infection.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

Characterization of the topology and functional domains of RKTG

2008 ◽  
Vol 414 (3) ◽  
pp. 399-406 ◽  
Author(s):  
Xiaolin Luo ◽  
Lin Feng ◽  
Xiaomeng Jiang ◽  
Fei Xiao ◽  
Zhenzhen Wang ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Structural Information ◽  
Transmembrane Protein ◽  
Cell Signalling ◽  
Amino Acid Sequences ◽  
Transmembrane Domains ◽  
Functional Domains ◽  
Golgi Localization ◽  
N Terminus

RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61–71 and 299–303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.

Molecular and Virulence Characterization of Newcastle Disease Virus in Fowls and Pigeons From Jammu, India

2019 ◽  
Author(s):  
Mehroon Chowdhary ◽  
Nawab Nashiruddullah ◽  
Parimal Roychoudhury ◽  
Altaf Bhat ◽  
Jafrin Ara Ahmed ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Newcastle Disease ◽  
Cleavage Site ◽  
Clinical Signs ◽  
Columba Livia ◽  
Clinical Samples ◽  
Protein Cleavage ◽  
Infectious Dose ◽  
Pcr Targeting

Occurrence of Newcastle disease (ND) and characterization of the Avian Paramyxovirus-1 (APMV-1) amongst backyard fowls (Gallus gallus domesticus) and pigeon (Columba livia domestica) was studied. ND was suspected in two fowl flocks (100% morbidity, 86% mortality) and six pigeon flocks (21.68% morbidity, 14.16% mortality) with respiratory and/or enteric clinical signs in fowls and predominantly neurological signs in pigeons was observed. Cloacal swabs, tissue lysates or infected allantoic fluid could haemagglutinate (HA) chicken erythrocytes and pigeon convalescent serum inhibited haemagglutination (antibody titres >1/16). A 534 bp product was amplified from clinical samples and infected allantoic fluids by Reverse Transcriptase PCR (RT-PCR) targeting a partial Fusion protein gene including its cleavage site. The fowl and pigeon isolate was found to be velogenic and lentogenic respectively by virulence characterization. Fowl isolate grown in chicken embryonated eggs (CEE) showed 10-8.68 Embryo Infectious Dose (EID50) with a Mean Death Time (MDT) of 51.43 h and the Intra-Cerebral Pathogenicity Index (ICPI) in day-old chicks was 1.51. Pigeon isolate had corresponding values of 10-7.20 EID50, MDT of 92.00 h and ICPI of 0.43. Deduced amino acid sequence at Fusion protein cleavage site showed 112R-R-R-K-R*F117 velogenic motif for the fowl isolate and 112G-R-Q-G-R*L117 lentogenic motif for the pigeon isolate. Both isolates were found to be 84.1% related phylogenetically, while fowl isolate clustered with genotype XIII, the pigeon isolate clustered with genotype II and vaccine strains of APMV-1. This is the first instance of APMV-1 characterization in the region with the possibility of a vaccine spill over in pigeons.

The Expression and Characterization of Gp85 Gene of Subgroup J Avian Leukosis Virus Associated with Hemangioma

2011 ◽  
Vol 343-344 ◽  
pp. 545-550
Author(s):  
Min Shi ◽  
Yan Lin ◽  
Yang Zhao ◽  
Ming Ping Guo ◽  
Luo Mei Sun ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Polyclonal Antibodies ◽  
Serum Antibody ◽  
Avian Leukosis Virus ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Gp85 Gene ◽  
High Level

The subtype of avian leukosis virus (ALV) was mainly determined by the gp85 glycoprotein. A subtype J ALV strain SCDY1 associated with hemangioma was isolated from grandparent breeding chicken and the highly antigenic region of its gp85 gene was amplified and expressed in Rosetta Escherichia coli using the pET-32a(+)vector. The fusion protein, which was expressed at a high level, was similar antigenically to the native gp85 protein as determined by Western blot assay using polyclonal antibodies against ALV-J strain. The fusion protein was also purified. This research lays a foundation for using this recombinant protein for development of indirect enzyme-linked immunosorbent assay (ELISA) for serum antibody detection or for production of monoclonal antibodies against prevalent ALV-J.

scholarly journals Mutant Analysis Reveals a Specific Requirement for Protein P30 in Mycoplasma pneumoniae Gliding Motility

2005 ◽  
Vol 187 (18) ◽  
pp. 6281-6289 ◽  
Author(s):  
Benjamin M. Hasselbring ◽  
Jarrat L. Jordan ◽  
Duncan C. Krause
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Cellular Polarity ◽  
Wild Type ◽  
Developmental Defects ◽  
C Terminus ◽  
Quantitative Examination

ABSTRACT The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.

Production and characterization of a single-chain variable fragment-alkaline phosphatase fusion protein for glycocholic acid detection in a one-step enzyme-linked immunosorbent assay

2018 ◽  
Vol 10 (22) ◽  
pp. 2629-2635 ◽  
Author(s):  
Xiping Cui ◽  
Qiyi He ◽  
Ding Shen ◽  
Zhengyun Jiang ◽  
Yingshan Chen ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Glycocholic Acid ◽  
One Step

One-step enzyme-linked immunosorbent assay for glycocholic acid based on single-chain variable fragment-alkaline phosphatase fusion protein.

Indirect enzyme-linked immunosorbent assay method based on Streptococcus agalactiae rSip-Pgk-FbsA fusion protein for detection of bovine mastitis

2017 ◽  
Vol 20 (2) ◽  
pp. 355-362
Author(s):  
Ri-E Bu ◽  
Jin-Liang Wang ◽  
Jin-Hua Wu ◽  
Gao-Wa Xilin ◽  
Jin-Long Chen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Immunosorbent Assay ◽  
Streptococcus Agalactiae ◽  
Membrane Surface ◽  
Bovine Mastitis ◽  
Assay Method ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Serum Dilution

Abstract Objective: The aim of this study was to establish a rapid and accurate method for the detection of the Streptococcus agalactiae antibody (SA-Ab) to determine the presence of the bovine mastitis (BM)-causative pathogen. Methods: The multi-subunit fusion protein rSip-Pgk-FbsA was prokaryotically expressed and purified. The triple activities of the membrane surface-associated proteins Sip, phosphoglycerate kinase (Pgk), and fibronectin (FbsA) were used as the diagnostic antigens to establish an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of SA-Ab in BM. Results: The optimal antigen coating concentration was 2 μg/mL, the optimal serum dilution was 1:160, and the optimal dilution of the enzyme-labeled secondary antibody was 1:6000. The sensitivity, specificity, and repeatability tests showed that the method established in this study had no cross-reaction with antibodies to Streptococcus pyogenes, Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis in the sera. The results of the sensitivity test showed that a positive result could be obtained even if the serum dilution reached 1:12,800, indicating the high sensitivity and good repeatability of the method. The positive coincidence rate of this method was 98.6%, which is higher than that of previous tests established with the Sip or Pgk mono-antigen fusion protein, respectively, demonstrating the relatively higher sensitivity of this newly established method. The detection rate for 389 clinical samples was 46.53%. Conclusions: The indirect ELISA method established in this study could provide a more accurate and reliable serological method for the rapid detection of S. agalactiae in cases of BM.

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