Biomedical subjects

N K Puri

Publications and source records attributed to N K Puri.

At least 19 recordsLinked to original sources

PubMed1996

Large scale purification process for recombinant NS1-OspA as a candidate vaccine for Lyme disease.

A purification process was developed for the manufacture of NS1-OspA as a candidate vaccine for Lyme disease. NS1-OspA was expressed as a soluble recombinant protein in E. coli from 50 and 200 liter fermentations. A multistep bench scale procedure, including detergent treatment, sonication, Q-Sepharose chromatography, dialysis, and SP-Sepharose chromatography, was used to effectively purify NS1-OspA from E. coli to > 90% purity with a 35% yield. This procedure was subsequently modified and specific steps eliminated to create a manufacturing scale process for the purification of NS1-OspA. Continuous flow centrifugation, mechanical lysis, QA-52 anion exchange chromatography, acid induced precipitation, and cross flow filtration gave a final purity of > 75%, a recovery of > 70%, and measured endotoxin levels of < 5 micrograms/mg of protein. Unique features of this process include using unclarified lysates and a single use (disposable) resin for the batch chromatography step. This simple, cost effective production process provides a recombinant protein with the yield and purity suitable for use as a subunit veterinary vaccine.

Animals↗

PubMed1995

Comparing the refolding and reoxidation of recombinant porcine growth hormone from a urea denatured state and from Escherichia coli inclusion bodies.

Overexpression of cloned genes in bacteria often leads to insoluble refractile body formation requiring solubilization and refolding to obtain biologically active proteins. A refolding pathway was established for a model protein, porcine growth hormone (PGH), yielding an appreciably high recovery of 85%. The conditions include the dilution of a urea, beta-mercaptoethanol (beta-ME) denatured PGH solution in a refolding environment containing 3.5 M urea and 10 mM beta-ME/HED at a 10:1 ratio at pH 9.1 and 0.5 mg/mL PGH. The intrinsic fluorescence-detected transition of PGH in urea gives 3.8 kcal/mol for the free energy of denaturation (delta GH2O) of PGH. The native-like conformation of PGH is dependent on disulfide bonds because reduced and carboxymethylated PGH is devoid of tertiary structure as assessed by intrinsic tryptophan fluorescence. Physical analysis of C-terminally truncated recombinant PGH indicated no significant difference in the free energy of denaturation of P-band in urea as full-length PGH. This suggests that the first disulfide, forming the large loop domain of PGH, provides a significantly greater contribution to the conformational stability of PGH than the second disulfide, which forms the carboxy-terminal small loop domain. The rate of formation of native structure during refolding was biphasic, with native structure identified by intrinsic fluorescence and hydrophobicity spectroscopy prior to disulfide bond formation. Thus "framework" intermediates are prerequisites for correct disulfide formation and tertiary folding of PGH. This study shows how a protein refolds, forms disulfides, and self-associates, which may be useful for examining the refolding of other recombinant proteins.

Animals↗

PubMed1994

A spectroscopic and equilibrium binding analysis of cationic detergent-protein interactions using soluble and insoluble recombinant porcine growth hormone.

Overexpression of cloned eukaryote genes in bacteria often leads to the formation of insoluble refractile bodies which require solubilization by harsh denaturants or detergents. We describe the conformational changes associated with the binding of a surfactant, cetyltrimethylammonium chloride (CTAC) to recombinant porcine growth hormone (PGH). The stoichiometry of binding by CTAC to the soluble and insoluble forms of recombinant PGH was also assessed. Optimum CTAC binding and protein solubilisation were obtained at 50 degrees C and at extreme pH. Increased ionic strength and changes in pH towards the isoelectric point of PGH (pH 6) decreased both the binding of CTAC and the efficiency of solubilising PGH from inclusion bodies. The positive charge on the quaternary ammonium head group of CTAC was found to be critical in the binding of CTAC to PGH and for the subsequent solubilisation of inclusion bodies. The binding of CTAC to the soluble form of PGH caused appreciable changes to the tertiary structure of the protein but did not significantly alter secondary structure, or cause complete unfolding. These observations help to explain earlier results which demonstrate that urea, guanidine hydrochloride and CTAC solubilized recombinant PGH molecules behave differently during in vitro refolding (Puri, N.K., Crivelli, E.C., Cardamone, M., Fiddes, R., Bertolini, J., Ninham, B. and Brandon, M.R. (1992) Biochem. J. 285, 871-879.).

Anilino Naphthalenesulfonates↗

PubMed1993

Characterization of a truncated form of recombinant porcine growth hormone generated in vitro during solubilization of inclusion bodies.

During the development of a novel solubilization procedure (1) for bacterial inclusion bodies (IB's) using the cationic surfactant cetyltrimethylammonium chloride (CTAC; (CH3)3-N(+)-C16H33Cl) significant proportions of an apparently truncated, lower molecular weight (MW) variant form of recombinant pig growth hormone (rPGH) were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to pig pituitary derived GH. The formation of this rPGH-like species, designated P-band, was found to occur in vitro during solubilization of IB's by CTAC and was dependent on pH and temperature of solubilization, but was not due directly to the use of CTAC, as purified soluble rPGH of the correct MW could not be converted to P-band by exposure to CTAC alone. The bacterial proteolysis suspected as being responsible for the in vitro formation of P-band could not be inhibited by the use of a "cocktail" of defined antiproteolytic agents but was inhibited by pH and temperature, and by solubilization of IB's in 5% SDS, 6 M gnHCl or 7.5 M urea. Detailed characterization of the structure of P-band by N-terminal amino acid sequencing, electrospray mass spectrometry, radioreceptor binding assay, peptide mapping, and C-terminal peptide sequencing confirmed that P-band was approximately 950 mass units smaller than normal rPGH and lacked eight C-terminal amino acids. A significant finding was that P-band is unable to bind to the pig liver-membrane GH receptor in a competitive radioreceptor assay. Analysis of the relative secondary and tertiary structure of P-band by circular dichroism spectra, intrinsic tryptophan-dependent fluorescence, and average surface hydrophobicity (2) suggested small but measurable changes to the overall structure of P-band relative to normal rPGH. Consequently, our results also suggest that the C-terminal portion of rPGH, including in particular the last eight amino acids, is of major importance in the binding of rPGH to the pig liver membrane GH receptor.

Amino Acid Sequence↗

PubMed1993

Comparison of conventional adjuvants and 'adjuvant-free' monoclonal antibody targeting for stimulating antibody responses against a conjugate of luteinizing hormone releasing hormone and avidin.

A conjugate of luteinizing hormone releasing hormone (LHRH) and avidin was used for immunizing mice and sheep. In mice, a range of adjuvants co-administered with the conjugate was shown to affect antibody titre and isotype. The commercially available Montanide and Ribi adjuvant emulsions, polyadenylic-polyuridylic acid, Quil A and Freund's complete adjuvant all produced high antibody titres to LHRH (reciprocal titre range 8000-20,000) and avidin (range 9000-35,000). Alhydrogel, Pluronic gel, Immunostim, DEAE-Dextran and muramyl dipeptide were weak to moderately effective (reciprocal titre ranges of 1300-5000 for LHRH and 3000-6000 for avidin, respectively). With Quil A, the response to avidin in mice was found to consist of roughly equal proportions of the IgG1, IgG2a and IgG2b isotypes. In contrast, the response to avidin using the other adjuvants and to LHRH consisted predominantly of the IgG1 isotype. An 'adjuvant-free' immunization strategy was attempted by targeting the LHRH-avidin conjugate to class II major histocompatibility complex (MHC) determinants on cells of the immune system. Using an immunoconjugate formed by linking LHRH-avidin to an antibody specific for mouse and sheep class II molecules, a twofold augmentation of titres relative to controls was obtained in mice. These antibodies were almost exclusively of the IgG1 isotype. In contrast, monoclonal antibody targeting in sheep resulted in a significantly enhanced immune response (reciprocal titres of 15,000 and 20,000 for LHRH and avidin, respectively) which was comparable to that achieved by immunizing with Quil A as adjuvant. Both the monoclonal antibody targeting and Quil A treatments tended to favour the production of antibody isotype IgG1 over IgG2.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

PubMed1992

Production of methionyl-minus ovine growth hormone in Escherichia coli and one-step purification.

The overproduction of ovine growth hormone (oGH) in Escherichia coli is described, achieved in part by alteration of the codon usage for nine of the first 15 amino acids (aa) of the mature hormone. Recombinant oGH (re-oGH), representing 12% of the total cellular protein, was isolated from inclusion bodies by solubilisation using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The hormone was refolded and subsequently purified to greater than 95% homogeneity in a single step using preparative reverse phase high performance liquid chromatography. The aa sequence analysis revealed that the N-terminus of the E. coli-derived polypeptide was identical to that of pituitary-derived oGH, and re-oGH displayed potent somatotropic activity in vivo.

Amino Acid Sequence↗

PubMed1992

Solubilization of growth hormone and other recombinant proteins from Escherichia coli inclusion bodies by using a cationic surfactant.

Recombinant pig growth hormone (rPGH) was solubilized from inclusion bodies by using the cationic surfactant cetyltrimethylammonium chloride (CTAC). The solubilizing action of CTAC appeared to be dependent on the presence of a positively charged head group, as a non-charged variant was inactive. Relatively low concentrations of CTAC were required for rapid solubilization, and protein-bound CTAC was easily removed by ion-exchange chromatography. Compared with solubilization and recovery of rPGH from inclusion bodies with 7.5 M-urea and 6 M-guanidinium chloride, the relative efficiency of solubilization was lower with CTAC. However, superior refolding efficiency resulted in final yields of purified rPGH being in the order of CTAC greater than urea greater than or equal to guanidinium chloride. Detailed comparison of the different rPGH preparations as well as pituitary-derived growth hormone by h.p.l.c., native PAGE, c.d. spectral analysis and radioreceptor-binding assay showed that the CTAC-derived rPGH was essentially indistinguishable from the urea and guanidinium chloride preparations. The CTAC-derived rPGH was of greater biopotency than pituitary-derived growth hormone. The advantages of CTAC over urea and guanidinium chloride for increasing recovery of monomeric rPGH by minimizing aggregation during refolding in vitro were also found with recombinant sheep interleukin-I beta and a sheep insulin-like growth factor II fusion protein. In addition, the bioactivity of the CTAC-derived recombinant interleukin-1 beta was approximately ten-fold greater than that of an equivalent amount obtained from urea and guanidinium chloride preparations. It is concluded that CTAC represents, in general, an excellent additional approach or a superior alternative to urea and in particular guanidinium chloride for solubilization and recovery of bioactive recombinant proteins from inclusion bodies.

Animals↗

PubMed1992

A relationship between the starting secondary structure of recombinant porcine growth hormone solubilised from inclusion bodies and the yield of native (monomeric) protein after in vitro refolding.

Recombinant porcine growth hormone (rPGH) was solubilised from inclusion bodies (IB's) using either 6 M guanidinium hydrochloride (GnHCl), 7.5 M urea or by a novel method using a cationic surfactant, cetyltrimethylammonium chloride (CTAC). Circular dichroism (CD) analysis of the secondary (2 degrees) structure of the urea- and GnHCl-solubilised rPGH showed the absence of alpha-helical content with the majority of the molecule existing in a 'random coil' structure. In contrast, the CTAC-solubilised rPGH displayed significant starting 2 degrees structure (10-15% alpha helix; 30-40% beta structure). The three rPGH preparations were refolded in vitro against weak urea. GnHCl or aqueous buffers, resulting in an average refolding efficiency of 50% native (monomeric) rPGH for CTAC solubilised IB's and only 20% for urea or GnHCl solubilised IB's. We conclude that the method of solubilisation of IB's and the resultant difference in the starting 2 degrees structure of rPGH, particularly alpha-helical content, is a major in vitro factor that apparently predetermines the aggregation/refolding behaviour rPGH irrespective of refolding environment.

Animals↗

PubMed1992

Spectrofluorimetric assessment of the surface hydrophobicity of proteins.

The equilibrium binding of the apolar fluorescent dye 1-anilinonaphthalene-8-sulphonate (ANS) to bacteriorhodopsin, BSA, chicken egg lysozyme, ovalbumin, porcine somatotrophin (PST) and bovine pancreatic ribonuclease (RNAase) was quantitatively evaluated using Scatchard- and Klotz-plot analyses. On the basis of the average association constant for ANS binding sites (Ka), the proteins could be ranked in order of surface hydrophobicity as: Bacteriorhodopsin greater than BSA greater than ovalbumin greater than PST greater than lysozyme greater than RNAase. The number of protein-ANS binding sites was determined as 54, 10, 3, 1, 2 and 1 respectively. The ANS-based assessment of the surface hydrophobicity of these proteins was generally in agreement with the average hydrophobicity based on amino acid sequence [Bigelow (1967) J. Theor. Biol. 16, 187-211], except for results with PST and ovalbumin. The proteins were also analysed by reversed-phase h.p.l.c. using C1 and C8 columns. There was no significant correlation between ANS and reversed-phase-h.p.l.c. assessment of hydrophobicity, with the results obtained by h.p.l.c. being dependent upon the column used. ANS-based measurement of surface hydrophobicity appears to be the most appropriate means for assessing proteins such as to reflect their overall three-dimensional structure in solution.

Chromatography, High Pressure Liquid↗

PubMed1991

Refolding of recombinant porcine growth hormone in a reducing environment limits in vitro aggregate formation.

Recombinant porcine growth hormone (rPGH) solubilized from bacterial inclusion bodies (IBs) using a cationic surfactant was oxidized to form disulphide bonds in a simple buffer solution containing 2-mercaptoethanol within an empirically derived optimal molar ratio of 2-mercaptoethanol:protein. A final yield of 55% monomeric rPGH was achieved at protein concentrations of up to 5 mg/ml without the need for removal of the 2-mercaptoethanol or the use of chaotrophic agents. In the absence of 2-mercaptoethanol only 15% monomeric rPGH was obtained, with the majority forming higher molecular weight aggregates. Using the procedure derived for porcine growth hormone, it may be possible to obtain high yields of native protein and overcome the need for using low protein concentrations and chaotrophic agents during in vitro refolding of other disulphide bonded recombinant proteins.

Animals↗

PubMed1991

A single step method for the solubilization and refolding of recombinant protein from E. coli inclusion bodies.

Expression of recombinant porcine growth hormone (rpGH) in E. coli cells resulted in the accumulation of the rpGH within inclusion bodies (IBs). The IBs were solubilized and the rpGH refolded in a single step using a cationic surfactant, N-cetyl pyridinium chloride (CPC; C21H38ClN) in the absence of reducing agents. No additional dialysis or rapid dilution steps of the solubilizing agent were required to obtain a 30% yield of refolded and oxidized rpGH monomer at protein concentrations of up to 15-20 mg/mL. In contrast, the refolding in vitro of rpGH in the absence of CPC resulted in the formation of significant amounts of higher molecular weight aggregate at the expense of the biologically active monomer. The fluorescence spectrum of the purified refolded rpGH was indistinguishable from that of biologically active, pituitary derived porcine GH and reverse-phase HPLC analysis of the purified rpGH showed similar retention times to that of pituitary GH. It is likely that the use of cetylpyridinium chloride is generally applicable to the simplified high yield recovery of biologically active recombinant proteins from IBs.

Animals↗

PubMed1987

N-terminal amino acid sequence analyses of the alpha and beta polypeptides from four distinct subsets of sheep major histocompatibility complex class II molecules.

Four monoclonal antibodies, SBU.II 28-1, 37-68, 38-27, and 42-20, each recognizing a distinct, non-overlapping subset of sheep class II molecules, were used to purify class II molecules from a single sheep. Four class II alpha subunits designated 28-1 alpha, 37-68 alpha, 42-20 alpha, and 38-27 alpha and five class II beta subunits designated 28-1 beta, 37-68 beta 1, 37-68 beta 2, 42-20 beta, and 38-27 beta were compared by N-terminal sequence analyses. Two distinct alpha subunits were identified; the 28-1 alpha, 37-68 alpha, and 42-20 alpha subunits all had identical N-terminal amino acids sequences, which exhibited about 75% homology with HLA-DR alpha and mouse E alpha polypeptides. In contrast, the 38-27 alpha sequence exhibited about 80% sequence homology with HLA-DQ alpha and mouse A alpha polypeptides. In general, sheep beta subunits displayed insufficient sequence homology to enable correlation with human beta-chain sequences; however, the 38-27 beta-chain sequence showed homology with the HLA-DQ beta sequence. The conserved sequence surrounding the site for N-linked glycosylation within human/mouse beta polypeptides (residues 19 to 21) was not present in sheep beta sequences and in contrast with the beta-chains of mouse and man, sheep beta polypeptides contained between 1 and 3 positionally variable cysteine residues (residues 13 to 15 inclusive). Individual sheep beta subunits exhibited extensive sequence heterogeneity and each consisted of a unique population of beta polypeptide species. At least 16 different beta polypeptide sequences were identified from a single sheep and the existence of no fewer than nine non-allelic beta genes was inferred from the sequence data. We have previously provided evidence suggesting that the sheep has multiple major histocompatibility complex class II alpha and beta genes related to those of all three HLA-D subregions. The present results suggest that a number of these genes encode HLA-DQ-like heterodimers and that a sheep DR-like alpha gene product is shared with the products of a large and heterogeneous sheep beta gene family.

Amino Acid Sequence↗

PubMed1987

A functional and biochemical analysis of bovine class II MHC antigens using monoclonal antibodies.

Monoclonal antibodies (MAbs) reacting with bovine (2) ovine (3), murine (1) or human (1) Class II MHC antigens were examined for reactivity with bovine peripheral blood leucocytes (PBL) and lymph node cells (LNC) by immunofluorescence, immunoprecipitation and the capacity to inhibit mixed lymphocyte responses (MLR), lectin- and antigen-induced blastogenesis. The 6 MAbs identified comparable percentages of Class II positive lymphocytes in PBL (40.8 to 54.2%) and LNC (6 to 11.5%) regardless of BoLA-A phenotype. Immunohistological staining of Class II MAb was localized principally to the lymphoid follicles in lymph nodes and to isolated epithelial reticular cells in the thymus. The anti-Class II MAb immunoprecipitated alpha- and beta- chains of 26-29K and 32-34K, respectively. These MAb inhibited proliferative responses in the MLR by between 25 and 74%, and diminished blastogenesis induced by specific antigens (purified protein derivative + PPD and ovalbumin) and B-lymphocyte mitogens (PPD, lipopolysaccharide and dextran sulphate) by between 45 and 75%, regardless of BoLA-A phenotype. In contrast, proliferation in response to concanavalin A and phytohaemagglutinin were unaffected by the anti- Class II MAb. Similarly these MAb did not affect lysis by cytotoxic T-lymphocytes, the activity of which was depressed by anti-Class I MAbs and monospecific alloantisera.

Animals↗

PubMed1987

Monoclonal antibodies to sheep MHC class I and class II molecules: biochemical characterization of three class I gene products and four distinct subpopulations of class II molecules.

The availability of a panel of monoclonal antibodies to sheep MHC class I and class II gene products has allowed for the first time an assessment of the relative complexity of the sheep MHC. By using four monoclonal antibodies to MHC class I, and seven monoclonal antibodies to MHC class II molecules together with one-dimensional SDS-PAGE, sequential immunoprecipitation and 2-dimensional gel analysis, three class I gene products and four distinct subsets of class II molecules have been identified. Sheep class I molecules showed heterogeneity on 2-dimensional gels and as in mouse and man, represented the products of at least three different non-allelic class I genes. Interestingly, the sheep beta 2 microglobulin molecule also displayed heterogeneity, consistent with either two primary gene products or allelic variation. Four sheep class II monoclonal antibodies identified distinct, non-overlapping subsets of sheep class II molecules of Mr 32-36 K (alpha chain) and 25-28 K (beta chain). These class II molecules were co-expressed on sheep B lymphocytes and represented the primary products of different sheep MHC class II genes. The class II molecules within three of these subsets displayed allelic polymorphism essentially restricted to their beta polypeptides, while the fourth subset of class II molecules showed allelic variation in both their alpha and beta polypeptides. The results of this study represent the first evidence for gene duplication and heterogeneity within the sheep MHC. The identification of three primary class I gene products and four distinct subsets of class II molecules suggests three class I loci and up to four distinct class II subregions within the sheep MHC. Potentially large numbers of allelic variants of these different gene products may be expressed in normal sheep.

Animals↗

PubMed1987

Biochemical and molecular analysis of sheep MHC class II molecules.

A panel of monoclonal antibodies was used for structural and immunodepletion analysis of sheep MHC class II molecules. The results indicate the antibodies recognize molecules of molecular weight 32-34,000 (alpha chain) and 26-28,000 (beta chain). Immunodepletion analysis indicates that the antibodies may recognize up to four distinct class II molecules some of which are structurally distinguishable using SDS-PAGE. Southern blot analysis using HLA-D region DR, DQ, DP, DO and DZ cDNA probes showed that a number of the cDNA probes hybridized specifically to sheep DNA indicating the presence of closely related genes in sheep. Together the results suggest that the sheep MHC class II region contains distinct MHC class II genes similar to those found in man.

Animals↗

PubMed1987

Monoclonal antibodies to sheep MHC class II molecules recognize all HLA-D or subsets of HLA-D region products.

Six murine monoclonal antibodies raised against sheep MHC class II molecules were analyzed for reactivity with HLA-D subregion products. All the antibodies reacted with human peripheral blood lymphocytes, monocytes, and B-lymphoblastoid cell lines homozygous for various HLA-DR specificities, suggesting that the antibodies recognized nonpolymorphic determinants on HLA class II molecules. SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analyses of molecules immunoprecipitated from 35S-methionine-labeled, DR-homozygous B-lymphoblastoid cell lines showed that the monoclonal antibodies precipitated typical class II molecules (Mr 32-34K and 25-29K). From comparison with antibodies of known HLA-D subregion specificity, two of the sheep antibodies appeared to react with the products of single HLA-D subregions, while another showed balanced reactivity with all HLA-D molecules. Antibody SBU.II 38-27 reacted exclusively with HLA-DQ molecules, antibody SBU.II 28-1 with HLA-DP molecules, and antibody SBU.II 49-1 with HLA-DR, -DQ, and -DP molecules. However, analysis of immunoprecipitates from surface-iodinated WT-49 cells (DR3 homozygous) using SBU.II 28-1 and the DP-specific monoclonal antibody B7/21, suggested that the two antibodies immunoprecipitated different alpha polypeptides. The two antibodies SBU.II 38-27 and 28-1 appear to be at least as specific as existing reagents, if not more so. As such, they are of value in their potential contribution to our understanding of the molecular characteristics and ultimately the functions of the HLA-DQ and -DP subregion products, as well as the identification/characterization of HLA-D equivalents in other species.

Adult↗

PubMed1987

Sheep MHC class II molecules. I. Immunochemical characterization.

The physicochemical features, biosynthesis and glycosylation of sheep class II molecules were investigated using a panel of seven monoclonal antibodies. The class II molecules recognized by different monoclonal antibodies could be differentiated using SDS-PAGE. Two monoclonal antibodies, SBU.II 42-20 and 49-1, reacted with dissociated sheep class II molecules and recognized epitopes on class II alpha and beta polypeptides, respectively. The structure of the sheep class II heterodimer differed from that of mouse and man in that it was unstable in the presence of 1% SDS at 20 degrees and, following reduction, sheep beta polypeptides displayed a marked increase in MW, resulting in the apparent co-migration of reduced alpha and beta polypeptides on SDS-PAGE. This phenomenon was not seen using sheep class II molecules synthesized in the presence of tunicamycin. Pulse-chase analyses of biosynthetically labelled sheep class II molecules suggested the rapid association and glycosylation of sheep class II alpha and beta polypeptides during synthesis. Both alpha and beta polypeptides of sheep class II molecules carried N-linked oligosaccharides of MW 6,000 and 3,000, respectively. However, unlike human class II oligosaccharides, these were exclusively of the complex or sialylated type.

Acetylglucosaminidase↗

PubMed1987

Sheep MHC class II molecules. II. Identification and characterization of four distinct subsets of sheep MHC class II molecules.

A panel of seven monoclonal antibodies, sequential immunoprecipitation and two-dimensional NEPHGE/SDS-PAGE analyses were used to identify and characterize subsets of sheep MHC class II molecules. Using sequential immunoprecipitation four distinct subsets of class II molecules were identified by the monoclonal antibodies SBU.II 28-1, 37-68, 38-27 and 42-20, while another monoclonal antibody, SBU.II 49-1, recognized all four subsets of class II molecules. These four subpopulations of sheep class II molecules displayed different two-dimensional gel profiles and, using splenocytes from four outbred sheep, the class II molecules recognized by SBU.II 28-1, 37-68 and 42-20 showed structurally detectable allelic polymorphism in their beta polypeptides, but no detectable variation in their alpha polypeptides. In contrast, the class II molecules recognized by SBU.II 38-27 showed allelic variation in both their alpha and beta polypeptides. Two-dimensional (2-D) gel analyses of non-glycosylated class II molecules immunoprecipitated by SBU.II 49-1 suggested that approximately 10-12 different class II molecules were expressed by a single sheep. The results of this study show that sheep express class II molecules that can be divided into four structurally and serologically distinct subsets, and provide additional evidence for the subdivision of the sheep MHC class II genetic region into at least three distinct subregions.

Animals↗