RESUMEN
Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.
Asunto(s)
Isótopos de Carbono , Clonación Molecular/métodos , Cianobacterias/genética , Girasa de ADN/biosíntesis , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/economía , Isótopos de Carbono/metabolismo , Girasa de ADN/química , Proteínas de Escherichia coli , Isótopos de Nitrógeno/economía , Isótopos de Nitrógeno/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Transformación BacterianaRESUMEN
Selectively-infective phage (SIP) is a novel methodology for the in vivo selection of interacting protein-ligand pairs. It consists of two components, (1) a phage particle made non-infective by replacing its N-terminal domains of geneIII protein (gIIIp) with a ligand-binding protein, and (2) an "adapter" molecule in which the ligand is linked to those N-terminal domains of gIIIp which are missing from the phage particle. Infectivity is restored when the displayed protein binds to the ligand and thereby attaches the missing N-terminal domains of gIIIp to the phage particle. Phage propagation is thus strictly dependent on the protein-ligand interaction. We have shown that the insertion of beta-lactamase into different positions of gIIIp, mimicking the insertion of a protein-ligand pair, led to highly infective phage particles. Any phages lacking the first N-terminal domain were not infective at all. In contrast, those lacking only the second N-terminal domain showed low infectivity irrespective of the presence or absence of the F-pilus on the recipient cell, which could be enhanced by addition of calcium. An anti-fluorescein scFv antibody and its antigen fluorescein were examined as a protein-ligand model system for SIP experiments. Adapter molecules, synthesized by chemical coupling of fluorescein to the purified N-terminal domains, were mixed with non-infective anti-fluorescein scFv-displaying phages. Infection events were strictly dependent on fluorescein being coupled to the N-terminal domains and showed a strong dependence on the adapter concentration. Up to 10(6) antigen-specific events could be obtained from 10(10) input phages, compared to only one antigen-independent event. Since no separation of binders and non-binders is necessary, SIP is promising as a rapid procedure to select for high affinity interactions.
Asunto(s)
Inovirus , Ligandos , Biblioteca de Péptidos , Proteínas/metabolismo , Cloruro de Calcio/farmacología , Proteínas de la Cápside , Proteínas de Unión al ADN/genética , Escherichia coli/virología , Fluoresceína , Fluoresceínas , Vectores Genéticos/genética , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Inovirus/genética , Inovirus/patogenicidad , Cloruro de Magnesio/farmacología , Unión Proteica , Proteínas/genética , Proteínas Virales de Fusión/genética , beta-Lactamasas/genéticaRESUMEN
To create a rapid system to test the effect of sequence changes on recombinant antibody binding, we have developed a procedure for producing functional scFv fragments in an Escherichia coli cell-free translation system. Functional antibodies with antigen-binding activity are obtained only if disulfide formation and rearrangement is allowed to take place during the translation reaction. The inclusion of protein disulfide isomerase (PDI) leads to a threefold increase in yield over that obtained in the presence of glutathione redox systems. DsbA had no such effect, indicating that disulfide shuffing, and not net formation, is the crucial yield-limiting step. The addition of the molecular chaperones DnaK and DnaJ increased the amount of soluble protein but not the amount of functional scFv, which appears to be limited entirely by correct disulfide formation. None of these factors significantly influenced total protein synthesis. In the presence of PDI, chaperones, reduced glutathione and oxidized glutathione, 50% of the scFv produced (about 8 micrograms/ml in only 15 min) could be recovered from immobilized antigen.
Asunto(s)
Formación de Anticuerpos/genética , Isomerasas/genética , Isomerasas/metabolismo , Chaperonas Moleculares/metabolismo , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Anticuerpos/genética , Western Blotting , Escherichia coli/genética , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Proteína Disulfuro Isomerasas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
We have developed a chloramphenicol resistant derivative of fd phage with which cognate pairs of antibodies and antigens can be selected. The phage genome encodes a fusion of single-chain antibody to the C-terminal domain of gIIIp, rendering the phage non-infective. The antigen fused to the N-terminal domains of gIIIp is encoded in the same phage genome. Antigen and antibody fusion interact with each other in the periplasm of the phage-producing cell, restoring infectivity. This system has a very low background and will allow simultaneous randomisation of antibody and antigen.
Asunto(s)
Complejo Antígeno-Anticuerpo/inmunología , Proteínas de Unión al ADN/inmunología , Inovirus/genética , Proteínas Virales de Fusión , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/genética , Proteínas de la Cápside , Cloranfenicol , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Vectores Genéticos , Genoma Viral , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Inovirus/inmunología , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
A systematic study has been performed on the relationship between linker length, relative orientation of variable domains, multimerization behaviour and antigen binding activity for single chain Fvs (scFvs) of the tumour-binding antibody B72.3. Thirteen scFv variants with linkers comprising up to six repeats of the motif Gly-Gly-Gly-Gly-Ser were studied. All these scFvs showed a tendency to form dimers or higher molecular weight species, and this tendency decreased with increasing linker length. The dimers and higher molecular weight forms may arise from head to tail intermolecular association of VH and VL domains. For each linker length, scFvs with the organization VL-linker-VH showed greater binding activity than those with the organization VH-linker-VL. In fact, for the latter organization only the variant with a 30 amino acid linker showed good binding activity, suggesting that (i) for B72.3 the C-terminus of VH or the N-terminus of VL makes a structural contribution to antigen binding, and (ii) shorter linkers interfere with this contribution. Antigen binding studies on scFvs should be interpreted with caution because of their tendency to multimerize. Such multimerization can be minimized by using linkers longer than those in common use.