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1.
J Pharm Biomed Anal ; 139: 143-147, 2017 May 30.
Article in English | MEDLINE | ID: mdl-28282600

ABSTRACT

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM197, Tetanus Toxoid and Diphtheria Toxoid.


Subject(s)
Chromatography, Gel/methods , Glycoconjugates/analysis , Polysaccharides, Bacterial/analysis , Salmonella typhi , Typhoid Fever , Typhoid-Paratyphoid Vaccines/analysis , Chromatography, High Pressure Liquid/methods , Glycoconjugates/therapeutic use , Humans , Typhoid Fever/prevention & control , Typhoid-Paratyphoid Vaccines/therapeutic use , Vaccines, Conjugate/analysis , Vaccines, Conjugate/therapeutic use
2.
Vaccine ; 30(5): 853-61, 2012 Jan 20.
Article in English | MEDLINE | ID: mdl-22172503

ABSTRACT

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.


Subject(s)
Citrobacter/immunology , Polysaccharides, Bacterial/isolation & purification , Typhoid-Paratyphoid Vaccines/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Citrobacter/chemistry , Humans , Polysaccharides, Bacterial/chemistry , Polysaccharides, Bacterial/metabolism , Technology, Pharmaceutical/methods , Typhoid-Paratyphoid Vaccines/chemistry , Typhoid-Paratyphoid Vaccines/metabolism , Vaccines, Conjugate/chemistry , Vaccines, Conjugate/metabolism
4.
Oncogene ; 19(17): 2147-54, 2000 Apr 20.
Article in English | MEDLINE | ID: mdl-10815806

ABSTRACT

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.


Subject(s)
Cell Transformation, Neoplastic/genetics , Genes, ras , Guanine Nucleotide Exchange Factors/genetics , ras Proteins/metabolism , Animals , Carcinogenicity Tests , Cell Division/genetics , Cell Line, Transformed , Down-Regulation , Female , Fibroblasts/pathology , Genes, Dominant , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Triphosphate/genetics , Guanosine Triphosphate/metabolism , Mice , Mice, Nude , Mutation, Missense , Signal Transduction , ras Proteins/genetics , ras-GRF1/genetics , ras-GRF1/metabolism
5.
J Immunol ; 158(8): 3881-7, 1997 Apr 15.
Article in English | MEDLINE | ID: mdl-9103457

ABSTRACT

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.


Subject(s)
Receptors, Interleukin-1/antagonists & inhibitors , Sialoglycoproteins/metabolism , Animals , Cell Line , Female , Humans , Interleukin 1 Receptor Antagonist Protein , Mice , Mice, Inbred C3H , Mutation , Protein Binding , Receptors, Interleukin-1/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sialoglycoproteins/genetics
6.
Protein Expr Purif ; 9(2): 219-27, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9056487

ABSTRACT

Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step.


Subject(s)
Bacillus subtilis/genetics , Bacillus subtilis/physiology , Receptors, Interleukin-1/antagonists & inhibitors , Recombinant Proteins/isolation & purification , Sialoglycoproteins/biosynthesis , Sialoglycoproteins/isolation & purification , Bacillus subtilis/chemistry , Cation Exchange Resins , Cell Division , Cell Fractionation , Cell Membrane Permeability/physiology , Cell-Free System , Humans , Interleukin 1 Receptor Antagonist Protein , Molecular Sequence Data , Recombinant Proteins/chemistry , Spores, Bacterial
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