Matty Feijlbrief

High level expression and secretion of truncated forms of herpes simplex virus type 1 and type 2 glycoprotein D by the methylotrophic yeast Pichia pastoris.

Herpes simplex virus type 1 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 be 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) (280-300mg/L induction medium) were produced. The yields of recombinant gD-1(1-254) and gD-1(1-254His) were lower: 20-36mg/L, and the yield of the gD-2(1-314) fragment was much lower: 6mg/L. SDS-PAGE analysis revealed multiple glycosylated species of the larger gD fragments, ranging in apparent molecular weight from 31 to 78kDa. The smaller gD-1(1-254) fragment appeared as two bands with molecular weights of 33 and 31kDa. All recombinant proteins produced by P. pastoris were recognized, as expected, by a panel of MAbs (A16, DL6, A18, DL11, HD1, ABDI, 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.

Synthesis, solution structure analysis and antibody binding of cyclic epitope peptides from glycoprotein D of Herpes simplex virus type I

Two cyclic peptides with a thioether bond have been synthesised corresponding to the 9-22 (9LKMADPNRFRGKDL(22)) sequence of glycoprotein D (gD-1) of Herpes simplex virus. The role of the secondary structure in protein-specific monoclonal antibody recognition was investigated. The sequence selected for this study comprises a strongly antigenic site adopting a beta-turn at residues 14Pro-(15)Asn. Thioether bond was formed between the free thiol group of cysteine or homocysteine inserted in position 11 and the chloroacetylated side chain of lysine in position 18. We report here the preparation of cyclic peptides containing Cys or Hcy in position 11, differing only in one methylene group. The linear precursor peptides were synthesised by Boc/Bzl strategy on MBHA resin, and the cyclisation was carried out in alkaline solution. The secondary structure of the peptides was studied by CD, FT-IR and NMR spectroscopy. The CD and FT-IR data have revealed fundamental changes in the solution conformation of the two compounds. The CH(2) group difference significantly resulted in the altered turn structure at the 12Ala and 13Asp as identified by NMR spectroscopy. The antibody binding properties of the cyclopeptides studied by gD-specific monoclonal antibody (A16) in direct and competition enzyme-linked immunosorbent assay (ELISA) were also not the same. We found that peptide LK[HcyADPNRFK]GKDL exhibited higher affinity to Mab A16 than peptide LK[CADPNRFK]GKDL, however, their reactivity was significantly lower compared to the linear ones. Our results clearly show the importance of secondary structure in an antibody binding and demonstrate that even a slight modification of the primary structure dramatically could influence the immune recognition of the synthetic antigens.

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

Detergents (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 (C10E5) 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 C10E5 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 C10E5 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.

Purification of the integral membrane glycoproteins D of Herpes simplex virus types 1 and 2, produced in the recombinant baculovirus expression system, by ion-exchange high-performance liquid chromatography

Selective elution of Sendai virus integral membrane proteins by ion-exchange high-performance liquid chromatography (HPIEC) using different detergent concentrations was reported before [S. Welling-Wester, M. Freijlbrief, D.G.A.M. Koedijk, M.A. Braaksma, B.R.K. Douma and G.W. Welling, J. Chromatogr., 646 (1993) 37]. In the present study this novel approach was applied to the purification of the integral membrane glycoprotein D of Herpes simplex virus type 1 and 2. The glycoproteins D of types 1 (gD-1) and 2 (gD-2) were cloned into the baculovirus expression system and produced in protein-free cultured insect cells. Detergent extracts of recombinant baculovirus-infected insect cells containing gD-1 or gD-2 were prepared using pentaethyleneglycol monodecyl ether, for extraction (final concentration 2%, w/v). The same detergent was used as additive in the elution buffers for HPIEC on a Mono Q HR 5/5 column. At low (0.005%) detergent concentration, most of the proteins present in the extract including part of gD were eluted with the sodium chloride gradient whereas a subsequent blank run using the same gradient at higher detergent concentration (0.1%) resulted in selective elution of pure gD.

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

Abstract Non-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 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 (C 10 E 5 and C 12 E 5 ) 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 C 10 E 5 and C 12 E 5 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.

COMPARISON OF DETERGENTS FOR EXTRACTION AND ION-EXCHANGE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF SENDAI VIRUS MEMBRANE-PROTEINS

The integral membrane proteins of Sendai virus, haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with a non-ionic and two zwitterionic detergents, i.e., pentaethylene glycol monolauryl ether (C12E5), lauryldimethylamine oxide (LDAO) and dodecyldimethylammoniopropane-1-sulphonate (SB12), respectively. The extracts were subjected to ion-exchange high-performance liquid chromatography (HPIEC) using 0.1% of the detergent in the eluent on four different columns (MA7Q, Zorbax BioSeries SAX, Mono Q and PL-SAX) with a quaternary amine as interacting ligand and with different pore sizes: non-porous and 30, 80 nm and 400 nm, respectively. The relative recoveries of protein were similar for all the columns. The highest recovery of HN and F protein and the best separation were obtained with C12E5. Analysis of HPIEC fractions with monoclonal antibodies directed against conformational epitopes showed that C12E5 had less effect on the conformation than the other two detergents.

REACTIVITY OF HUMAN SERA WITH OVERLAPPING SYNTHETIC PEPTIDES OF HERPES-SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN-D

SummaryThirty eight human sera, seropositive for herpes simplex virus (HSV) and 56 human sera, seronegative for HSV by immunofluorescence and by ELISA, were investigated for reactivity with a series of overlapping synthetic peptides of HSV type 1 glycoprotein D (gD-1). Thirty four out of the 38 human sera positive for HSV reacted with peptides located between residues 300 and 369; the HSV-negative sera reacted with six of the gD-1 peptides, but with none of the peptides within residues 300 to 369.

Analogues of peptide 9–21 of glycoprotein D of herpes simplex virus and their binding to group VII monoclonal antibodies

SummarySeveral analogues of the amino acid sequence of peptide 9–21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were synthesized and investigated for reactivity with different group VII monoclonal antibodies, Mabs LP14, ID3, 170, HD4, A16, EII-24 and EV-10, in a competition enzyme-linked immunosorbent assay (ELISA). Replacement of Arg at position 16 by His resulted in a loss of binding with the group VII Mabs. Substitution of Pro at residue 14 by Leu gave a reduced binding for a number of Mabs and loss of binding for Mab A16. Substitution of Lys at position 10 by Glu gave reduced binding for three out of the seven Mabs. In addition substitutions of Met at position 11 by norleucine and oxidized Met were studied. The boundaries of the epitope cluster were mapped by studying synthetic variants of peptide 9–21 which were shorter either at the C-terminus or at the N-terminus, or both. Peptide 10–18 and peptide 9–17 were the shortest peptides, which were still reactive with the group VII Mabs. Mab HD4 requires the N-terminus of peptide 9–21 for effective binding, while for binding of other Mabs contribution of the residues in the C-terminal part of this peptide is more important.

Synthesis and Antibody Recognition of Cyclic Epitope Peptides, Together with Their Dimer and Conjugated Derivatives Based on Residues 9-22 of Herpes Simplex Virus Type 1 Glycoprotein D

The synthesis of new cyclic peptides comprising the 9-22 epitope (9)LKMADPNRFRGKDL(22) sequence derived from HSV gD-1 is reported. In addition, we describe procedures for the preparation of cyclic peptide dimers and conjugates with an oligotuftsin derivative carrier. The binding of a monoclonal antibody, Mab A16, to the synthesized compounds was determined by enzyme-linked immunosorbent assay. It was demonstrated that cyclization decreased the binding activity of the antibody to the epitope. However, dimerization and conjugation could significantly increase the binding capacity of the cyclic epitope peptides. The attachment site in dimers and conjugates, as well as the topology of the construct, had a significant influence on the antibody recognition, while replacement of Met in position 11 by Nle had no marked effect.

Synthetic mini-antibodies in biosensors

Biorecognition may play an important role in real-time detection of proteins in biosensors. Recognition may be achieved by antibodies or fragments thereof. Antibodies can specifically recognize proteins by six different Complementarity Determining Regions (CDRs), which are positioned on the hypervariable segments of the heavy and light chains of the antibody molecule. Synthetic peptides of individual CDRs may also be able to recognize a protein antigen and are called mini-antibodies. We intend to use antilysozyme peptides and antiherpes gD (glycoprotein of herpes simpex virus) peptides in a surface plasmon resonance biosensor. CDRs were selected from the amino acid sequences of monoclonal antibodies (mAbs) against lysozyme and gD. The selection of antilysozyme peptides was facilitated by the availibility of tertiary structure information on three different antibody-lysozyme complexes. These mAbs (D1.3, HyHEL 5 and Gloop 1-5) are directed against different epitopes of lysozyme. Binding was studied by immunoaffinity chromatography and by ELISA. With ELISA three different lysozyme binding peptides were found i.e. H2D1.3 (15 a.a. residues), (H1+H2)D1.3 (36 a.a. residues) and H2HyHEL 5 (17 a.a residues). Binding and specificity of these peptides and two Gloop2 peptides (L3Gloop2, 10 a.a residues and H2Gloop2, 13 a.a residues)1,2 was also tested by immunoaffinity chromatography. Antilysozyme peptide columns could retard lysozyme compared with a control column. It was also possible to purify lysozyme from a mixture of different proteins. Binding studies with mini-antibodies against herpes gD are in progress.