16 research outputs found
Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography
No full textDetergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant baculovirus. The highest yield was obtained with the two alkyl carboxybetaine detergents (N-dodecyl-N,N-dimethylammonio)undecanoate [DDMAU, critical micelle concentration (CMC)=0.13 mM] and (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC=4.3 mM). Therefore these zwitterionic detergents were used as additives to the elution buffers in ion-exchange high-performance liquid chromatography (HPIEC) to purify gD-1 of HSV-1 from the extracts. The non-ionic detergent pentaethyleneglycol monodecyl ether (C(10)E(5)) that was used in earlier studies [R.A. Damhof, M. Feijlbrief, S. Welling-Wester: G,W Welling, J. Chromatogr. A, 676 (1994) 43] was used for comparison. Two columns were used, Mono Q and Resource Q, at 1 and 5 ml/min flow-rates, respectively. The results show that the detergents DDMAU and C(10)E(5) are superior to DDMAB, when the detergents were used as additives to the elution buffers at 0.2% (w/v). With 0.2% DDMAB in the eluent, purification of HSV gD-1 was not possible. Detergents with a high CMC may be less suitable as additives in elution buffers. HPIEC at flow-rates of 1 and at 5 ml/min showed satisfactory results. At 5 ml/min HSV gD-1 was mainly concentrated in two eluent fractions. The highest recovery of gD-1 was obtained either by chromatography of a C(10)E(5) extract using a Mono Q column at a flow-rate of 1 ml/min or by chromatography of a DDMAU extract using a Resource Q column at a flow-rate of 5 ml/min. (C) 1998 Elsevier Science B.V. All rights reserved.</p
Detergent extraction of herpes simplex virus type 1 glycoprotein D by zwitterionic and non-ionic detergents and purification by ion-exchange high-performance liquid chromatography
No full textDetergents (surfactants) are the key reagents in the extraction and purification of integral membrane proteins. Zwitterionic and non-ionic detergents were used for the extraction of recombinant glycoprotein D (gD-1) of herpes simplex virus type 1 (HSV-1) from insect cells infected with recombinant baculovirus. The highest yield was obtained with the two alkyl carboxybetaine detergents (N-dodecyl-N,N-dimethylammonio)undecanoate [DDMAU, critical micelle concentration (CMC)=0.13 mM] and (N-dodecyl-N,N-dimethylammonio)butyrate (DDMAB, CMC=4.3 mM). Therefore these zwitterionic detergents were used as additives to the elution buffers in ion-exchange high-performance liquid chromatography (HPIEC) to purify gD-1 of HSV-1 from the extracts. The non-ionic detergent pentaethyleneglycol monodecyl ether (C(10)E(5)) that was used in earlier studies [R.A. Damhof, M. Feijlbrief, S. Welling-Wester: G,W Welling, J. Chromatogr. A, 676 (1994) 43] was used for comparison. Two columns were used, Mono Q and Resource Q, at 1 and 5 ml/min flow-rates, respectively. The results show that the detergents DDMAU and C(10)E(5) are superior to DDMAB, when the detergents were used as additives to the elution buffers at 0.2% (w/v). With 0.2% DDMAB in the eluent, purification of HSV gD-1 was not possible. Detergents with a high CMC may be less suitable as additives in elution buffers. HPIEC at flow-rates of 1 and at 5 ml/min showed satisfactory results. At 5 ml/min HSV gD-1 was mainly concentrated in two eluent fractions. The highest recovery of gD-1 was obtained either by chromatography of a C(10)E(5) extract using a Mono Q column at a flow-rate of 1 ml/min or by chromatography of a DDMAU extract using a Resource Q column at a flow-rate of 5 ml/min. (C) 1998 Elsevier Science B.V. All rights reserved
Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance
No full textThe interaction between mAb A16 and glycoprorein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, 4sp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide.</p
Kinetic analysis of synthetic analogues of linear-epitope peptides of glycoprotein D of herpes simplex virus type 1 by surface plasmon resonance
No full textThe interaction between mAb A16 and glycoprorein D (gD) of herpes simplex virus type 1 was analyzed by studying the kinetics of binding with a surface-plasmon-resonance biosensor. mAb A16 belongs to group VII antibodies, which recognize residues 11-19 of gD. In a previous study, three critical residues, 4sp13, Arg16 and Phe17, of this epitope were identified by screening a phage display library that contained a random 15-amino-acid insert with the antibody. The contribution to binding of these residues in the motif DXXRF was further analyzed by an amino-acid-replacement study of the epitope gD-(9-19)-peptide and of a gD-(9-19)-peptide mimotope, previously obtained by screening the phage display library. Amino acid residues of the motif were replaced by a neutral amino acid residue, an amino acid residue with opposite charge and a corresponding D-amino acid residue. Kinetic parameters of peptide analogues were determined with a surface plasmon-resonance biosensor. The kinetic parameters of the peptide analogues were compared with the kinetic parameters of the interaction between mAb A16 and the epitope gD-(9-19)-peptide. The minimal size of the gD epitope for mAb A16 was also determined in this study. The kinetic constants of the resulting gD-(11-17)-peptide were found to be similar to those of entire gD. The kinetic analysis precisely defined the epitope on gD for mAb A16 to residues 11-17, identified Arg16 as an essential residue and suggested that Asp13 and Phe17 are mainly involved in stabilization of the secondary structure of the peptide
Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins:a wash step with a low concentration of EDTA
No full textImmobilised metal-ion affinity chromatography (IMAC) is widely used for the purification of recombinant proteins in which a poly-histidine tag is introduced. However, other proteins may also bind to IMAC columns. We describe the use of a washing buffer with a low concentration of EDTA (0.5 mM) for the removal of proteins without histidine tag from IMAC columns. Four histidine-tagged recombinant proteins/protein complexes were purified to homogeneity from cell culture medium of insect cells by using an EDTA washing buffer. The presence of a low concentration of EDTA in washing buffers during IMAC may have a general application in the purification of histidine-tagged proteins. (C) 2001 Elsevier Science B.V. All rights reserved.</p
Effect of different amounts of the non-ionic detergents C10E5 and C12E5 present in eluents for ion-exchange high-performance liquid chromatography of integral membrane proteins of Sendai virus
No full textNon-ionic detergents (0.03-0.5%) are used as additives to the eluents when integral membrane proteins are subjected to ion-exchange high-performance liquid chromatography (HPIEC). It is not known whether this concentration should bear some relation to the critical micelle concentration (CMC) of a detergent (the concentration at which micelles begin to form) or that only the amount of detergent is of importance in order to maintain the membrane proteins in solution. This was investigated with a detergent extract of Sendai virus which contains two integral membrane proteins, the fusion protein and the haemagglutinin-neuraminidase protein. Two polyoxyethylene alkyl ethers (C10E5 and C12E5) were used both for extraction (2% final concentration) and as additives in the elution buffers for HPIEC on Mono Q with ''classical'' HPLC and the micro-HPLC Smart System (Pharmacia-LKB). The CMCs of the two non-ionic detergents C10E5 and C12E5 are 0.026 and 0.002%, respectively. Concentrations below and above the CMC were used in the eluent. The results showed that the concentration of the detergent should be 2-26 times the CMC in order to avoid aggregation. The integral membrane proteins of Sendai virus remain on the column when the detergent concentration is less than 0.026-0.05%, independent of the CMC of the detergent. This may be utilized in HPIEC strategies: at low detergent concentration, hydrophilic proteins are eluted with the salt gradient and a subsequent blank run with the same gradient at higher detergent concentrations results in elution of the integral membrane proteins
Zenobia’s Baths in Palmyra (Syria): An Assessment
No full textInternational audienceThe Roman baths of Palmyra were excavated by Syrian archaeologists between 1953 and 1975, in the center of the historic city, between the monumental arch and the tetrapylon. In 2009, within the frame of the project “Balneorient” (http://balneorient.hypotheses.org/), the French Institute in the Near East (Ifpo) and the Syrian Direction of Antiquities and Museums (DGAM) decided to complete the study of this building, visited by thousands of tourists since its discovery, but still unpublished. The beginning of the Syrian civil war in 2011 prevented us from completing this work. After almost 7 years of conflict, the situation is more dire than ever, and many major monuments of Palmyra had disappeared. Considering the risk of destruction to Zenobia’s Baths, and the necessity to raise awareness of this threatened heritage, we have decided to present the uncomplete results of its study.The paper will present the monumental remains of the buildings (hypocausted rooms, ornamented basilica, porticoed palaestra and natatio, Diocletian’spropyleon), the results of the early excavations, and will then propose an architectural reconstruction ofthe building at each period of its long chronology (i.e. from the 2nd c. AD to the 6th c. AD). The links between the complex urban history of Palmyra and the social/political significance of public baths in Late Roman Near East will then be explored. The results of this Palmyrene case study will finally be comparedto ongoing works by the author on other remoted oriental Roman baths (Karnak and Petra), at the margin of the empire
Heraqla, between the dream of Harun Al Rashid and the nightmare of the looters [Elektronisk resurs]
No full textThe archaeological site of Heraqla in Raqqa represents a particular example of the Abbasid architecture; the Caliph Harun Al Rashid started reconstructing it as a commemorative monument but after his death it was never completed. The Directorate General of Antiquities and Museums DGAM excavated in the site, constructed furnace to produce bricks similar to the ancient material for restorations and, most importantly, built storehouses to deposit the archaeological items revealed during many decades of local, international and joint excavations. These storehouses were inventoried once in 2003 by the author as a preliminary inventory to integrate them in a sophisticated database later. Regretfully, in 2013, some 100, armed looters robbed the storehouses. The present paper is the only documentation ever completed on Heraclea’s storehouses and aims to raise awareness about their importance, to encourage researchers to create a database of the looted objects to identify them, facilitate their recovering and restitution if they appear on the black market of antiquities, or at least prevent their circulation freely through sharing the database with INTERPOL and other authorities involved in fighting against illicit trafficking
Heraqla, between the dream of Harun Al Rashid and the nightmare of the looters
No full textThe archaeological site of Heraqla in Raqqa represents a particular example of the Abbasid architecture; the Caliph Harun Al Rashid started reconstructing it as a commemorative monument but after his death it was never completed. The Directorate General of Antiquities and Museums DGAM excavated in the site, constructed furnace to produce bricks similar to the ancient material for restorations and, most importantly, built storehouses to deposit the archaeological items revealed during many decades of local, international and joint excavations. These storehouses were inventoried once in 2003 by the author as a preliminary inventory to integrate them in a sophisticated database later. Regretfully, in 2013, some 100, armed looters robbed the storehouses. The present paper is the only documentation ever completed on Heraclea’s storehouses and aims to raise awareness about their importance, to encourage researchers to create a database of the looted objects to identify them, facilitate their recovering and restitution if they appear on the black market of antiquities, or at least prevent their circulation freely through sharing the database with INTERPOL and other authorities involved in fighting against illicit trafficking
High level expression and secretion of truncated forms of herpes simplex virus type I and type 2 glycoprotein D by the methylotrophic yeast Pichia pastoris
No full textHerpes simplex virus type I and 2 (HSV-1 and -2) glycoproteins D (gD-1 and gD-2) play a role in the entry of the virus into the host cell. Availability of substantial amounts of these proteins, or large fragments thereof. will he needed to allow studies at the molecular level. We studied the potency of the Pichia pastoris yeast expression system to produce Soluble forms of gD. The DNA sequences encoding the extracellular domains of gD {amino acids 1-314 (gD-1((1-314))) and amino acids 1 254 (gD-1((1-254))) of gD-1 and amino acids 1-314 of gD-2 (gD-2((1-314)))} were cloned into the P. pastoris yeast expression vector pPIC9, Two truncated forms of gD-1 were fitted with a His tail (designated as gD-1((1-314His)) and gD-1((1-254His))) to facilitate their purification. Large amounts of gD-1((1-314)) and gD-1((1-314His)) (280-300 mg/L induction medium) were produced, The yields of recombinant gD-1((1-254)) and gD-1((1-254His)) were lower: 20-36 mg/L. and the yield of the gD-2((1-314)) fragment was much lower: 6 mg/L. SDS PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78 kDa. The smaller gD-1((1-254)) fragment appeared as two bands with molecular weights of 33 and 31 kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6. A18. DL11, HD1, ABD1. and AP7). In addition, we showed that gD-1((1-314)), gD-2((1-314)), and gD-1((1-254His)) were able to interfere with binding of HSV to susceptible cells. These results indicate that the conformations of the recombinant proteins closely resemble those of native gD. (C) 2002 Elsevier Science (USA). All rights reserved.</p
