scholarly journals Expression, Purification, and Characterization of Ras Protein (BmRas1) fromBombyx mori

2012 ◽  
Vol 2012 ◽  
pp. 1-8
Author(s):  
Yanping Quan ◽  
Guangqiang Liu ◽  
Wei Yu ◽  
Zuoming Nie ◽  
Jian Chen ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Malpighian Tubule ◽  
Silk Gland ◽  
Random Coil ◽  
Intrinsic Activity ◽  
Ras Signaling ◽  
Reading Frame ◽  
Cellular Signal

The Ras subfamily is the member of small G proteins superfamily involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation, and survival.Bombyx moriRas-like protein (BmRas1) may belong to the Ras subfamily. It contained an H-N-K-Ras-like domain. The BmRas1 mRNA consisted of 1459 bp. The open reading frame contained 579 bp, encoding 192 amino acids. The protein had such secondary structures asα-helices, extended strand, and random coil. BmRas1 was expressed successfully inE. coliBL21. The recombinant protein was purified with metal-chelating affinity chromatography. The GTPase activity of purified protein was determined by FeSO4-(NH4)2MoO4assay. The results showed that purified recombinant protein had intrinsic activity of GTPase. High titer polyclonal antibodies were generated by New Zealand rabbit immunized with purified protein. The gene expression features of BmRas1 at different stages and in different organs of the fifth instar larvae were analyzed by Western blot. The results showed that BmRas1 was expressed highly in three development stages including egg, pupae, and adult, but low expression in larva. BmRas1 was expressed in these tissues including head, malpighian tubule, genital gland, and silk gland. The purified recombinant protein would be utilized to further function studies of BmRas1.

scholarly journals Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1421
Author(s):  
Yong Yang ◽  
Jinkai Zang ◽  
Shiqi Xu ◽  
Xueyang Zhang ◽  
Sule Yuan ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Neutralizing Antibodies ◽  
Wild Type Strain ◽  
Immune Escape ◽  
High Titer ◽  
Prototype Strain ◽  
Vaccine Antigen ◽  
Broadly Neutralizing Antibodies ◽  
Continued Use ◽  
Further Development

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate.

Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection

2014 ◽  
Vol 196 ◽  
pp. 7-14 ◽  
Author(s):  
Reetika Kapoor ◽  
Bikash Mandal ◽  
Prabir Kumar Paul ◽  
Phaneendra Chigurupati ◽  
Rakesh Kumar Jain
Keyword(s):  
Recombinant Protein ◽  
Polyclonal Antibodies ◽  
Virus Detection ◽  
Multiple Virus

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

An electrogenic Na+- HCO 3 − cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain

2000 ◽  
Vol 278 (6) ◽  
pp. C1200-C1211 ◽  
Author(s):  
Mark O. Bevensee ◽  
Bernhard M. Schmitt ◽  
Inyeong Choi ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Rat Brain ◽  
Cortical Neurons ◽  
Polyclonal Antibodies ◽  
Amino Acid Level ◽  
Cdna Libraries ◽  
Human Pancreas ◽  
Opposite Pattern ◽  
Reading Frame ◽  
Expression Studies ◽  
Rapid Amplification

We screened rat brain cDNA libraries and used 5′ rapid amplification of cDNA ends to clone two electrogenic Na+-[Formula: see text] cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3′ end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (αrb1NBC) and rb2NBC (αrb2NBC). αrb1NBC labels an ∼130-kDa protein predominantly from kidney, and αrb2NBC labels an ∼130-kDa protein predominantly from brain. αrb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; αrb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO2/10 mM [Formula: see text] to Xenopus oocytes injected with rb2NBC cRNA causes 1) pHi to recover from the initial CO2-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na+ reverses the pHi increase and elicits a rapid depolarization. In the presence of 450 μM DIDS, removing external Na+ has no effect on pHi and elicits a small hyperpolarization. The rate of the pHidecrease elicited by removing Na+ is insensitive to removing external Cl−. Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

scholarly journals Identification of a Fibronectin-Binding Protein from Staphylococcus epidermidis

2002 ◽  
Vol 70 (12) ◽  
pp. 6805-6810 ◽  
Author(s):  
Rachel J. Williams ◽  
Brian Henderson ◽  
Lindsay J. Sharp ◽  
Sean P. Nair
Keyword(s):  
Recombinant Protein ◽  
Staphylococcus Epidermidis ◽  
Binding Protein ◽  
Phage Display Library ◽  
Binding Domain ◽  
Genomic Research ◽  
Gene Encoding ◽  
Reading Frame ◽  
Fibronectin Binding Protein ◽  
Fibronectin Binding

ABSTRACT Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

A Novel Bioadhesive Protein of Silk Filaments Spun Underwater by Caddisfly Larvae

2009 ◽  
Vol 79-82 ◽  
pp. 1631-1634 ◽  
Author(s):  
Yu Jun Wang ◽  
Kazumi Sanai ◽  
Masao Nakagaki
Keyword(s):  
Amino Acids ◽  
Silk Gland ◽  
Molecular Surface ◽  
Common Amino Acid ◽  
Reading Frame ◽  
Underwater Adhesion ◽  
Charged Amino Acids ◽  
Disulphide Bond Formation ◽  
Caddisfly Larvae ◽  
Order Of Magnitude

Aquatic Larvae of Stenopsychid caddisfly (Stenopsychie marmorata) survive by attaching its catching nets at the bottom of the rocks in the flowing water. It was hypothesized that S. marmorata larva connects small pebbles by producing both silk-like protein and strong adhesive protein simultaneously. A 98 kDa protein（Smap-98k）was identified as an adhesive component by constructing a silk gland –specific cDNA library of S. marmorata. The cDNA sequence of Smap-98k was 2,679 bp long and encoding a 893 amino acids–long open reading frame (ORF) in which the first 19 residues are predict to be the signal peptide. The alignment of the Cys residues indicated the primary structure of this protein to consist of 15 degenerated repeats, each about 50 residues long and contains 6 conserved Cys residues. The Smap-98k was characterized by an abundance of Cys residues and charged amino acids with epidermal growth factor-like (EGF-like) structure. The most common amino acid of this protein was Cys (11.98%), with Pro (9.91%) and Glu (9.26%) following order of magnitude. Cys was assumed to play a role in maintaining the topology of charged amino acids on the molecular surface by intramolecular disulphide-bond formation. The gene was expressed specially in the silk gland similarity to the major silk proteins such like heavy fibroin (H-fibroin) and Light fibroin (L-fibroin) of S. marmorata larvae. The sequence of the protein showed certain homology to the silk-185 kDa of Chironomus pallidivittatus (Midge) which also spin silk underwater. The characterizations of abundance of Cys residues and charged amino acids also shared by Megabalanus rosa cement protein (Mrcp-20k ) and Mytilus galloprovincialis foot protein 2 (Mgfp 2) which both were produced in the marine environment. Although the similarity among Smap-98k, Mrcp-20k and Mgfp 2 sequences were very low, the functional relationship in underwater adhesion of these proteins should be noted.

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

1990 ◽  
Vol 36 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Apurba K. Bhattacharjee ◽  
Elizabeth E. Moran ◽  
Wendell D. Zollinger
Keyword(s):  
Bactericidal Activity ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Rabbit Serum ◽  
Meningococcal Vaccine ◽  
The Poor ◽  
Group B ◽  
Antibody Levels ◽  
Sepharose 4B ◽  
Immune Rabbit

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28 400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000–2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine. Key words: anti-Lip, antibodies, bactericidal, Neisseria, Lip.

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