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Biomedical subjects

H Gram

Publications and source records attributed to H Gram.

29 records · Page 2Linked to original sources
PubMed1994

A novel approach for high level production of a recombinant human parathyroid hormone fragment in Escherichia coli.

We describe a novel approach to the production in E. coli of a peptide fragment derived from the human parathyroid hormone (hPTH). The first 38 amino acids of hPTH were fused at the amino terminus to a derivative of the bacteriophage T4-encoded gp55 protein, and were expressed in the E. coli cytoplasm in inclusion bodies at levels exceeding 50% of the total cell protein. Solubilization and subsequent incubation of the inclusion bodies in dilute hydrochloric acid facilitated the cleavage of an acid-labile aspartyl-prolyl peptide bond engineered into the fusion protein, thus releasing the hormone fragment directly from the inclusion body preparation. The amino-terminal prolyl-prolyl dipeptide-extension was subsequently removed by treatment with Lactococcus lactis dipeptidyl peptidase IV which was overexpressed in E. coli and purified to near homogeneity from the cytosol of the recombinant bacteria. In pilot-scale fermentations, more than 80 mg of pure hPTH(1-38) were isolated per liter of bacterial culture, with an overall yield of 35%. This process is suitable for scale-up, is cost effective, and by employing recombinant dipeptidyl peptidase IV, should be widely and directly applicable to the manufacturing of peptides of pharmaceutical interest.

Amino Acid Sequence↗
PubMed1993

Phage display as a rapid gene expression system: production of bioactive cytokine-phage and generation of neutralizing monoclonal antibodies.

Proteins, such as hormones, enzymes, or antibody binding sites, can be expressed in an active conformation on the surface of filamentous bacteriophage. Although the phage display technology was originally developed for binding studies, we demonstrate here that this technique can rapidly provide cytokines for studies of biological activity and for raising neutralizing monoclonal antibodies. A phage M13-based cloning vector was constructed that facilitated the expression of human interleukin 3 (hIL-3) on the phage surface. The recombinant phage could stimulate the growth of the hIL-3 dependent cell line M-07, providing evidence for the display of hIL-3 in an active form. Injection of recombinant phage into mice provoked an immune response to hIL-3, and neutralizing monoclonal antibodies directed against native hIL-3 could be established from these mice with a high frequency.

Amino Acid Sequence↗
PubMed1992

In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library.

We have used a combinatorial immunoglobulin library approach to obtain monoclonal antibodies from nonimmune adult mice, thereby establishing the principles of (i) accessing naive combinatorial antibody libraries for predetermined specificities and (ii) increasing the affinity of the selected antibody binding sites by random mutagenesis. A combinatorial Fab library expressing immunoglobulin mu and kappa light-chain fragments on the surface of filamentous phage was prepared from bone marrow of nonimmunized, adult BALB/c mice with the multivalent display vector pComb8. Phage displaying low affinity Fabs (binding constants, 10(4)-10(5) M-1) binding to a progesterone-bovine serum albumin conjugate were isolated from the library. Random mutagenesis of the heavy- and light-chain variable regions expressed in the mono-valent phage display vector pComb3 was performed by error-prone PCR, and subsequently clones with improved affinity for the hapten conjugate were selected. We demonstrate that antibodies with desirable characteristics from a nonimmune source may be selected and affinity maturation may be achieved by using the twin vectors pComb8 and pComb3, thus opening the route to obtaining specific antibodies from a generic library and bypassing immunization.

Amino Acid Sequence↗
PubMed1992

High-level expression of a human immunoglobulin gamma 1 transgene depends on switch region sequences.

We describe that chimeric mouse-human immunoglobulin heavy chain (IgH) genes lacking a switch region and controlled by an IgH promoter and the intronic enhancer are only weakly expressed in transgenic mice. Insertion of part of the human C gamma 1 or murine Cmu switch region into the major intron of the chimeric IgH gene results in a 10(2)-to 10(3)-fold increase in transgene expression. Analysis of B cell hybridoma clones from transgenic mice suggests that switch sequences influence IgH transgene expression at the cellular level. However, the effect of switch region sequences on IgH gene expression observed in vivo is not apparent in transfected B cell lines. These results indicate that switch region sequences which are located proximal to the constant part of the gene and which are normally present in a rearranged IgH gene after class switching represent a novel type of regulatory element that plays a critical role in IgH gene expression in vivo.

Animals↗
PubMed1989

Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions.

A chimeric human-mouse anti-T lymphocyte mAb (CHT2; SDZ 214-380) has been constructed by cloning the variable region exons of both the L and H chains from the murine hybridoma RFT2 which have CD7 specificity and reactivity with a 40-kDa Ag. The variable L chain exon was joined to the human C kappa, and the variable H chain exon was joined to the human IgG1 region exon encoding the human allotype nGlm(z), nGlm(a). The gene constructs were introduced by electroporation into SP2/0, a non-Ig-producing murine myeloma. The identical tissue reactivity of the newly made CHT2 and the original murine RFT2 mAb (CD7) was confirmed by blocking experiments as well as by immunohistology and flow cytometry. Because this new mAb may have clinical use, the CD7 Ag expression of T lineage cells has also been quantitated in double and triple immunofluorescence assays in combinations with mAb to restricted forms of leukocyte common Ag that designate unprimed (CD45R+) and primed T lymphocyte populations (UCHL1+). CHT2 shows very strong reactivity with large thymic blast cells and cortical thymocytes from which T-ALL originates. Strong staining is seen on CD45R+ unprimed "virgin" T lymphocytes, whereas the expression on UCHL1+ primed "memory" cell types is weaker unless these cells are reactivated by mitogens or Ag. Thus CHT2 may spare a substantial population of resting memory T cells which is relevant to its potential therapeutic use. In addition the chimeric antibody had a greater in vitro antibody dependent cytotoxicity and a prolonged half-life (4.2 to 5.0 days) in Rhesus monkeys.

Amino Acid Sequence↗
PubMed1988

Complete protein sequences of the variable regions of the cloned heavy and light chains of a human anti-cytomegalovirus antibody reveal a striking similarity to human monoclonal rheumatoid factors of the Wa idiotypic family.

The complete amino acid and nucleotide sequences of the variable regions of the heavy and light polypeptide chains of a human neutralizing IgGl anti-cytomegalovirus (CMV) antibody reveal a striking homology to IgM rheumatoid factors (RFs) of the Wa idiotypic family. The anti-CMV antibody and Wa RFs have in common VKIIIb, JKl, and VHIa gene segments but use different DH and JH gene segments. The anti-CMV antibody does not have RF activity and does not express the Wa idiotype. The Wa RFs do not have anti-CMV activity. A subset of Wa RFs, however, and the anti-CMV antibody do share several idiotypes on the VHIa and VKIIIb polypeptides. Since there are major differences in the antigen binding characteristics and some of the other expressed idiotypes, these data suggest that the D and J region amino acids are crucial to such specificities. Although the use of such highly homologous gene segments in different immune responses is well-documented in murine systems, these data represent the first such example in the human.

Amino Acid Sequence↗
PubMed1986

The alpha-glucosyltransferases of bacteriophages T2, T4 and T6. A comparison of their primary structures.

With the aim of comparing the primary structures of gene products coded for by T-even bacteriophages we constructed clone libraries of the DNAs of bacteriophages T2 and T6. Using hybrid M13 phages carrying the gene for the T4-coded alpha-glucosyl transferase (alpha gt) we isolated corresponding T2 and T6 clones. The nucleotide sequences of the three alpha gt genes and the amino acid sequences derived were compared. The differences between the genes and their products are discussed in terms of structure, function and evolutionary aspects.

Amino Acid Sequence↗
PubMed1985

T4-induced alpha- and beta-glucosyltransferase: cloning of the genes and a comparison of their products based on sequencing data.

Bacteriophage T4 alpha- and beta-glucosyltransferases link glucosyl units to the 5-HMdC residues of its DNA. The monoglucosyl group in alpha-linkage predominates over the one in beta linkage. Having recently reported on the nucleotide sequence of gene alpha gt (1) we now determined the nucleotide sequence of gene beta gt. The genes were each cloned on a high expression vector under the control of the lambda pL promoter. After thermo-induction the proteins were isolated and purified to homogeneity. To verify that the translational starting sites and the proposed reading frames are effective in vivo the sequence of the first 31 amino acid residues from gp alpha gt and the first 30 amino acid residues from gp beta gt were determined by Edman degradation. The primary structures of the two proteins seem to have only limited structural similarities. The results are discussed comparing secondary structure predictions and homologies with other proteins from the protein sequence database of the Protein Identification Resource.

Amino Acid Sequence↗
PubMed1985

Genes 55, alpha gt, 47 and 46 of bacteriophage T4: the genomic organization as deduced by sequence analysis.

The nucleotide sequence of T4 genes 55, alpha gt, 47 and 46 was determined by a combination of 'classical' procedures and a shotgun approach. Small DNA fragments generated by frequent cleavage with restriction enzymes or by sonication of restriction fragments were cloned in phage M13 vectors and sequenced by the dideoxy method. The positions of the genes were determined by marker rescue between the corresponding T4 amber mutants and the cloned T4 DNA fragments used in the sequencing experiments. The sequence gives an insight into the organization of this 7.1-kb early region of the T4 genome and shows that genetically 'silent' portions within this region are not void of genetic information.

Base Sequence↗