ABSTRACT
Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Hemoglobins/metabolism , Oxygen/metabolism , Allosteric Regulation , Genetic Variation , Hemoglobins/chemistry , Hemoglobins/genetics , Humans , Hydrogen-Ion Concentration , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , TransfectionABSTRACT
The proliferative T cell responses to poly(GluLysTyr) (GLT) and poly(GLULysPhe) (GLPhe) are restricted by the E alpha E beta class II MHC molecule (E) in most responded strains. Some nonresponder strains that carry responder E beta, but cannot express cell surface E molecules, can complement with other nonresponder strains that provide the missing E alpha chain needed for the expression of E molecules and for responsiveness to GLT and GLPhe. Here another type of complementation is described between two E-nonexpressor haplotypes, H-2f and H-2s, which result in E-nonexpressor F1 hybrids, which are responders to GLT. The restriction element involved in this response is an Af/As hybrid molecule. The data support the hypothesis that conformational determinants resulting from the free association of alpha and beta chains in heterozygotes can increase the immune potential of the individual.
Subject(s)
Histocompatibility Antigens Class II/immunology , Peptides/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/immunology , Cross Reactions , Genetic Complementation Test , H-2 Antigens/immunology , Histocompatibility Antigens Class II/genetics , Lymphocyte Activation , Mice , Mice, Inbred Strains/immunology , Polymers , Protein Conformation , Protein MultimerizationABSTRACT
The immunogenicity of the four sequential polymers of alpha-L-amino acids referred to below was studied in inbred strains of mice. The responses were linked to the H-2 haplotype, and the Ir genes controlling the responses were mapped to the IA region. The responses were restricted to two haplotypes as follows: (Ala-Tyr-Glu-Gly)n and (Ala-Glu-Tyr-Gly)n - H-2k; (Glu-Ala-Tyr-Gly)n - H-2b; (Glu-Tyr-Ala-Gly)n - H-2k and b. The absence of reciprocal crossreactions among all of these polymers, plus the sequential polymers (Tyr-Ala-Glu-Gly)n and (Tyr-Glu-Ala-Gly)n at both the antibody and T-cell levels, indicated that each of these six polymers was indeed structurally unique.
Subject(s)
Histocompatibility Antigens Class II/genetics , Major Histocompatibility Complex , Mice, Inbred Strains/immunology , Peptides/immunology , Receptors, Antigen, T-Cell/genetics , Amino Acid Sequence , Animals , Antibody Formation , Genetic Linkage , Haplotypes , Histocompatibility Antigens Class II/immunology , Mice , Mice, Inbred Strains/genetics , Molecular Sequence Data , Receptors, Antigen, T-Cell/isolation & purification , T-Lymphocytes/immunologyABSTRACT
The murine immune response patterns to (Phe-Glu-Ala-Gly)n and (Phe-Ala-Glu-Gly)n differ from those of the tyrosine analogues (Tyr-Glu-Ala-Gly)n and (Tyr-Ala-Glu-Gly)n. (Phe-Glu-Ala-Gly)n was not immunogenetic in inbred, congenic or recombinant strains of mice. (Phe-Ala-Glu-Gly)n was immunogenic only in mice having f alleles in the IA subregion of the H-2 complex. Reciprocal in vitro cross reactions were noted with T cells from mice of H-2f that respond to either (Phe-Ala-Glu-Gly)n or (Tyr-Ala-Glu-Gly)n. T cells from mice of H-2b, which are responders to (Tyr-Glu-Ala-Gly)n, could not be stimulated with the nonimmunogenic (Phe-Glu-Ala-Gly)n. These results support other studies showing that for any polymer to elicit cross T-cell proliferative responses it must be (a) structurally related to the homologous polymer and (b) immunogenic in the mouse strain whose T cells are being challenged in vitro. Explanations are offered for these statements.