RESUMEN
An efficient and inexpensive laboratory approach for the generation and the purification of polyclonal antibodies to human antigen CD34 was developed. It was shown that cloned refolded and purified from Escherichia coli recombinant extracellular fragment of CD34 antigen retained immunogenic determinants of cell-surface expressed CD34. Immunization of mice with unglycosylated truncated recombinant protein elicit polyclonal antibodies specific for the native human antigen CD34. The antibodies generated are applicable for phenotyping of CD34+ cells using immunocytochemistry and flow cytometry assays.
Asunto(s)
Anticuerpos/aislamiento & purificación , Antígenos CD34/inmunología , Animales , Anticuerpos/inmunología , Antígenos CD34/genética , Western Blotting , Línea Celular Tumoral , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Femenino , Vectores Genéticos , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
One of the most important problems in biotechnology today is development of simple and cheap techniques for protein purification. This is why inteins and protein splicing phenomena are of a great interest for protein purification. Modification of inteins by affinity tags permits to use general methods for protein purification. Following autocatalytic excision of tagged intein from protein allows to obtain pure protein without formyl-methionin residue on its N-terminus. New method for two step protein purification on the base of protein splicing phenomena has been created. Using modified intein Mxe GyrA extra pure human growth hormone with minimal lost and with native N-terminus was obtained.
Asunto(s)
Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/aislamiento & purificación , Inteínas/genética , Empalme de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Biotecnología/métodos , Hormona de Crecimiento Humana/biosíntesis , Humanos , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
A panel of single-chain antibodies (ScFv's--single-chain Fv-antibodies) against recombinant human interferon beta 1b (rhIFN-beta1b) has been obtained from immune and naive combinatorial cDNA libraries of the mouse variable immunoglobulin genes. ScFv's were expressed into Escherichia coli cells. For producers isolated from the immune library a difference in production yield of ScFv's in periplasm and incubation medium as well as their expression stability in passages and storage stability have been demonstrated. After sequencing of target DNA the multiple alignment and structural analysis of ScFv's sequences with different primary structures were carried out and significant difference in both complementarity-determining (CDR) and framework (FR) regions of their variable domains has been shown. For the ScFv's isolated from the immune library, specificity of their binding with native and denatured rhIFN-beta1b in ELISA and Western-blotting as well as their high storage stability have been shown. The affinity constants for each representatives of the ScFv's panel were in the range from 1.96 x 10(-8) to 1.69 x 10(-9) M.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Interferón beta/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Western Blotting , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Biblioteca de Genes , Humanos , Immunoblotting , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Interferon beta-1b , RatonesRESUMEN
Protein splicing is a post-translational autocatalytic process that results in excision of internal peptide (intein) from a precursor protein and the ligation of the flanking protein sequences (exteins). High specificity of the intein-mediated excision of protein precursors allows the use of protein splicing in biotechnology. This work was aimed at the obtaining of human growth hormone with a native N-terminus in E. coli. Chimerical protein consisting of a short N-terminal peptide, Mxe GyrA intein and human growth hormone was created. During the translation formyl-methionine modified N-terminal peptide should have been removed by splicing. Intein was shown to mediate the cleavage of exteins, but their subsequent ligation was not observed. That allowed the preparation of human growth hormone with a native N-terminus. The effect of various factors on cleavage efficiency was studied. The most efficient cleavage of chimeric protein (60-80%) was achieved in the presence of inductor (100 mM beta-mercaptoethanol) upon the incubation for 4-6 days.
Asunto(s)
Escherichia coli/genética , Hormona del Crecimiento/biosíntesis , Inteínas/genética , Ingeniería de Proteínas , Empalme de Proteína/genética , Proteínas Recombinantes de Fusión/biosíntesis , Catálisis , Hormona del Crecimiento/genética , Humanos , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Escherichia coli/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Interferón-alfa/inmunología , Animales , Anticuerpos Monoclonales/genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Humanos , Fragmentos de Inmunoglobulinas/genética , Cuerpos de Inclusión , Interferón alfa-2 , Ratones , Plásmidos , Renaturación de Proteína , Proteínas RecombinantesRESUMEN
The gene of ScFv-CBD-fusion protein has been designed using the DNA sequences encoding of single-chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) and cellulose-binding domain (CBD) from Clostridium thermocellum cellulosome. Biosynthesis of ScFv-CBD utilizing high-productive Escherichia coli system was carried out and the accumulation of target protein in bacterial inclusion bodies was shown. After the purification of the inclusion bodies and their subsequent in vitro refolding the soluble ScFv-CBD-fusion protein was directly immobilized on cellulose by bioaffinity coupling. The possibility to obtain the preparative quantities of ScFv-CBD in biologically-active form using different refolding schemes was accurately investigated in the paper. The general applicability of biologically immobilized ScFv-CBD-fusion proteins for affinity purification of recombinant IFN-alpha2b is shown.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Celulosa/metabolismo , Cromatografía de Afinidad/métodos , Fragmentos de Inmunoglobulinas , Interferón-alfa/inmunología , Proteínas Recombinantes de Fusión , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Cuerpos de Inclusión , Interferón alfa-2 , Plásmidos , Reacción en Cadena de la Polimerasa , Renaturación de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas RecombinantesRESUMEN
A combinatorial library of single-chain antibodies (ScFv) from mice immunized with human alpha2b interferon (hIFN-alpha2b) was constructed. For obtaining of phage display antibodies the DNA fragments of ScFv were cloned into phagemid vector pCANTAB-5E and rescued from Escherichia coli cells by infection with bacteriophage M13. Bacterial clones synthesizing specific ScFv against hIFN-alpha2b were isolated by several rounds of affinity selection of phage library. After isolation and affinity purification of ScFv-IFN from bacteria cells a high ability to binding of the hIFN-alpha2b has been shown. The sequencing of isolated ScFv DNA and analysis of the data obtained have been carried out. The data of expression stability of obtained E. coli producers such as some features of ScFv expression are also discussed.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Fragmentos de Inmunoglobulinas/biosíntesis , Interferón-alfa/inmunología , Biblioteca de Péptidos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Bacteriófagos/genética , Clonación Molecular , Dermatoglifia del ADN , Cartilla de ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/inmunología , Interferón alfa-2 , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
Transfer of human apoAI gene, within the molecular construction which provides its expression, to the liver of adult and aged rats resulted in the appearance of human protein in their blood, and was accompanied by changes in the content of high-density lipoproteins, as well as by the shifts in their protein and lipid composition. Administration of the human ApoAI gene was followed by changes of the vasoactive effects of HDL. Gene implantation is capable of enhancing the direct vasodilatory effects of HDL in old animals, being weakened by ageing, even against the background of normal age changes in the vascular wall tone.