Sytske Welling-Wester

PREDICTION OF SEQUENTIAL ANTIGENIC REGIONS IN PROTEINS

Prediction of antigenic regions in a protein will be helpful for a rational approach to the synthesis of peptides which may elicit antibodies reactive with the intact protein. Earlier methods are based on the assumption that antigenic regions are primarily hydrophilic regions at the surface of the protein molecule. The method presented here is based on the amino acid composition of known antigenic regions in 20 proteins which is compared with that of 314 proteins [(1978) Atlas of Protein Sequence and Structure, vol. 5, suppl. 3, 363‐373]. Antigenicity values were derived from the differences between the two data sets. The method was applied to bovine ribonuclease, the B‐subunit of cholera toxin and herpes simplex virus type 1 glycoprotein D. There was a good correlation between the predicted regions and previously determined antigenic regions.

THE INFLUENCE OF PH AND IONIC-STRENGTH ON THE COATING OF PEPTIDES OF HERPES-SIMPLEX VIRUS TYPE-1 IN AN ENZYME-LINKED IMMUNOSORBENT-ASSAY

Rabbits were immunized with synthetic peptides of herpes simplex virus type 1 glycoproteins, coupled to a carrier protein with glutaraldehyde. Antibodies directed against the peptides were determined in an enzyme-linked immunosorbent assay (ELISA). Either free peptides or peptides coupled with glutaraldehyde to another carrier protein than the one used for immunization were used as the coating antigen. When conjugated peptides were used as the coat, it was necessary in some instances to correct the antibody titers for a substantial amount of antibody activity against glutaraldehyde. When free peptides were used, optimal coating conditions with regard to pH and ionic strength had to be determined, since some peptides failed to coat under standard conditions, at pH 9.6. The results show that some peptides needed stringent pH conditions while others could be coated in a broad pH range. The addition of 0.6 M NaCl had a favorable effect on peptide coating.

THE INFLUENCE OF DIFFERENT ADJUVANTS ON THE IMMUNE-RESPONSE TO A SYNTHETIC PEPTIDE COMPRISING AMINO-ACID RESIDUES 9-21 OF HERPES-SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN-D

The immuno-modulating properties of different adjuvant systems on the murine humoral and cellular immune response to a synthetic peptide comprising amino acid residues 9-21 of glycoprotein D of herpes simplex virus type 1 (HSV-1) were investigated. For immunization, the peptide was conjugated to ovalbumin or bovine serum albumin by glutaraldehyde and the adjuvants used in this study were Freunds complete adjuvant (FCA), aluminium hydroxide, the Ribi adjuvant system (RAS) and two non-ionic block polymer surfactants, viz. L101 and 31R1, in oil in water emulsions. High anti-peptide antibody titers were obtained after immunization with FCA, aluminium hydroxide, RAS and L101. All adjuvants, except RAS, stimulated the induction of delayed type hypersensitivity obtained after immunization with peptide 9-21 coupled to ovalbumin and elicited by injection of purified HSV-1 virions in the footpad. Challenge with a lethal dose of HSV-1 showed that mice immunized with peptide 9-21 coupled to ovalbumin in combination with FCA, RAS and L101, respectively, were significantly protected. Although immunization with peptide 9-21 coupled to ovalbumin combined with aluminium hydroxide stimulated induction of delayed type hypersensitivity, no significant protective immunity against the challenge was generated.

Purification of detergent-extracted Sendai virus proteins by reversed-phase high-performance liquid chromatography.

Sendai virus envelope proteins were isolated by reversed-phase high-performance liquid chromatography. The F (F1 and F2, connected by disulphide bonds), M and HN proteins were extracted from purified Sendai virions with Triton X-100. After removal of the detergent from the extract with Amberlite XAD-2 and reduction of the proteins, separation was performed on a small (10-nm) and on a larger (30-nm) pore size C18 column. Proteins were eluted with a gradient of an ethanol 1-butanol mixture in 12 mM hydrochloric acid. On the 10-nm pore size column, F2 was completely recovered in pure form, whereas the recoveries of the other proteins were low (5-25%). Similar results were obtained with the 30-nm pore size column, except for protein F1 of which the yield was higher (50%).

ISOLATION OF DETERGENT-EXTRACTED SENDAI VIRUS PROTEINS BY GEL-FILTRATION, ION-EXCHANGE AND REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AND THE EFFECT ON IMMUNOLOGICAL ACTIVITY

Virus envelope proteins were isolated from Triton X-100 extracts of purified Sendai virions by gel-filtration, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC). The fusion protein F, the matrix protein M and the tetrameric and dimeric form of the HN protein were isolated by gel-filtration HPLC with a solvent containing 0.1% sodium dodecyl sulphate. HN and F were also isolated by ion-exchange HPLC with 0.1% Triton X-100 in the eluent. Reversed-phase HPLC was performed on a C1 column with acetonitrile as the organic solvent. Especially the F1 and F2 component of the fusion protein F were obtained in pure form. The immunological activity of the proteins after HPLC was determined with an enzyme-linked immunosorbent assay (ELISA). After gel-filtration and ion-exchange HPLC, proteins still reacted with antiserum to the intact virus while proteins purified by reversed-phase HPLC did not react.

Synthetic antibody fragment as ligand in immunoaffinity chromatography

The possibility that a fragment of an antibody molecule may interact with a protein antigen was tested by studying the binding properties of a thirteen-residue synthetic peptide with an amino acid sequence similar to part of a hypervariable segment of a monoclonal antibody directed against lysozyme. Affinity adsorbents were prepared with this peptide and with non-related peptides as ligand. Non-specific interactions could be abolished by washing the column with 0.05 M sodium thiocyanate in 20 mM tris-HCl (pH 7.4). Lysozyme was only bound to the antilysozyme adsorbent and could be eluted with 1 M sodium thiocyanate. The results show that immunoaffinity chromatography with synthetic peptide ligands which mimic the antigen-binding site may be a useful tool in the selective purification of proteins.

Immobilised metal-ion affinity chromatography purification of histidine-tagged recombinant proteins: a wash step with a low concentration of EDTA

Immobilised 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.

Combined size-exclusion and reversed-phase high-performance liquid chromatography of a detergent extract of sendai virus

Virus envelope proteins obtained by Triton X-100 extraction of Sendai virions were purified to a high degree by a combination of high-performance liquid chromatography (HPLC) methods. Size-exclusion HPLC on a TSK 4000 PW column with several concentrations of acetonitrile or ethanol-1-butanol in 0.1% hydrochloric acid as eluent was used as the first chromatographic step. Peak fractions were diluted in water and further fractionated on reversed-phase columns (TMS-250 or Vydac 218 TP). Size-exclusion HPLC with 45% acetonitrile in 0.1% hydrochloric acid, combined with reversed-phase HPLC on either column, was most suitable for obtaining highly purified F2 protein. Antibodies obtained after injection of this protein were reactive with the intact virus.

COLUMN LIQUID-CHROMATOGRAPHY OF INTEGRAL MEMBRANE-PROTEINS

Abstract Biological membranes have as a major function the compartmentation of biological processes in cells and organelles. They consist of a bilayer of phospholipid molecules in which proteins are embedded. These integral membrane proteins, which cross the bilayer once or several times, generally have a higher than average hydrophobicity and tend to aggregate. Detergents are needed to remove integral membrane proteins from the lipid bilayer and they have to be present during further chromatographic purification. Predominantly, four modes of HPLC have been used alone or in combination for the puridication of integral membrane proteins. These are based on differences of proteins in size (size-exclusion chromatography, SEC), electrostatic interaction (ion-exchange chromatography, IEC), bioaffinity (bioaffinity chromatography, BAC) and hydrophobic interaction (reversed-phase chromatography, RPC, and hydrophobic-interaction chromatography, HIC). SEC, IEC, BAC and HIC are used under relatively mild conditions, and buffer systems generally contain a non-ionic detergent. RPC generally has a denaturing effect on the protein and should preferably be used for the purification of integral membrane proteins smaller than 50 kD.

SIZE-EXCLUSION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF SENDAI VIRUS MEMBRANE-PROTEINS IN DIFFERENT DETERGENTS - A COMPARISON OF DIFFERENT COLUMNS

Four column packings for size-exclusion high-performance liquid chromatography (HPLC) of proteins with particle sizes from 3 to 13 micron were compared, using 0.1% sodium dodecyl sulphate in the solvent. Their suitability for the purification of hydrophobic membrane proteins was studied with Sendai virus proteins as a model. The calibration curves of the two 13-micron column packings were linear up to high molecular weights. In contrast to this, large proteins (greater than 100-150 kD) were eluted later than expected from the 3- and 6-micron packings. Peak capacities for proteins larger than 20 kD ranged from 4.7 to 5.5. Therefore, purification of complex mixtures of membrane proteins will often require rechromatography by a different mode of HPLC. Non-ionic detergents are suitable for further ion-exchange chromatography. The effect of addition of 0.1% of five non-ionic detergents (three gluco-methylalkanamide detergents, octylglucoside, and decyl-polyethyleneglycol-300) to the solvent was investigated and decyl-polyethyleneglycol-300 was found to be most suitable. Size-exclusion HPLC with this detergent resulted in the separation of micelles of three different sizes, of which the larger two contained exclusively the Sendai virus F protein.