The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.
A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary-based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.
Interleukin-3 (IL-3) is a cytokine which stimulates the proliferation and differentiation of hematopoietic progenitors into multiple cell lineages. The 1H, 15N, and 13C NMR resonances of a recombinant human IL-3 variant (SC-65369) have been assigned using two- and three-dimensional NMR techniques on uniformly 13C/15N-enriched protein. Five helical segments (residues 16-26, 42-50, 55-65, 73-82, and 104-120) and three reverse turns (residues 51-54, 68-71, and 87-90) were identified from the pattern of sequential NOE connectivities, NH(i)-C alpha H(i) scalar coupling constants (3JNH alpha), amide hydrogen exchange data, and the deviation of 13C alpha, 13C beta, 13CO, and C alpha H chemical shifts from random-coil values. Long-range NOEs indicate that the global folding pattern of human IL-3 is a four-helical bundle with an up-up-down-down arrangement of helices that is similar to that of other members of the cytokine family, such as granulocyte-macrophage colony stimulating factor (GM-CSF). A fifth short helix (helix A', residues 42-50) is located in the loop connecting the first and second helices. The absence of helix A' in the corresponding structures of GM-CSF and interleukin-5 suggests that it may be important for recognition of IL-3 by its receptor. The existence of at least two forms of the protein that differ in local conformation was implied from the observation of a limited set of doubled resonances in which each doublet partner had a similar pattern of short-, medium-, and long-range NOEs. The majority of the doubled resonances were close in sequence or space to a proline-rich sequence, which suggested that the different conformational forms of SC-65369 may be caused by slow cis-trans isomerization of proline peptide bonds.
Steady-state kinetic analysis of human renin demonstrates the histidine proximal to the substrate scissile peptide bond contributes to the unique specificity and pH dependence of this aspartyl protease. Recombinant human renal renin purified from mammalian cell culture appears to be indistinguishable from renin isolated from human kidney with respect to specific activity (1000 Goldblatt units/mg). Recombinant renin contains carbohydrate covalently attached to asparagines at positions 5 and 75 (renin numbering) and disulfide linkages at Cys-51/Cys-58, Cys-217/Cys-221, and Cys-259/Cys-296. Renin pH dependence was evaluated between pH 4.0 and 8.0 by using a synthetic substrate identical with the amino terminus of porcine angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu*Leu-Val-Tyr-Ser, where the asterisk indicates the scissile peptide bond and the proximal histidine is in italics) and an analogous tetradecapeptide where the proximal histidine was substituted with glutamine. Comparison of the pH profiles shows the catalytic efficiency (V/Km) and maximal velocity (V) of renin are greater above pH 6.5 with the substrate containing histidine proximal to the scissile peptide bond, but below pH 5.0 these parameters are greater with the glutamine substrate analogue. Solvent isotope effects show that proton transfer contributes to the rate-limiting step in catalysis with both substrates and that the proximal histidine does not serve as a base in the catalytic mechanism. Molecular modeling indicates the substrate histidine could hydrogen bond to Asp-226 of the enzyme (renin numbering), thus perturbing the ionization of the catalytic aspartyl groups (Asp-38 and Asp-226).(ABSTRACT TRUNCATED AT 250 WORDS)
Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild-type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.
The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.
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