RESUMEN
A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein. In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle. The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively. Human kappa constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-kappa antibody. This display system may complement currently available bacterial selection techniques.
Asunto(s)
Bacteriófagos/fisiología , Proteínas de Unión al ADN/metabolismo , Biblioteca de Péptidos , Proteínas/análisis , Receptores de Estrógenos/metabolismo , Bacteriófagos/genética , Secuencia de Bases , Cadherinas/análisis , Cadherinas/genética , Cadherinas/metabolismo , Ligasas de Carbono-Carbono/análisis , Ligasas de Carbono-Carbono/genética , Ligasas de Carbono-Carbono/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/análisisRESUMEN
The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition.
Asunto(s)
Bacterias/aislamiento & purificación , Etanolaminas , Reacción en Cadena de la Polimerasa/métodos , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Microbiología del Suelo , Ácidos Alcanesulfónicos , Animales , Bacterias/genética , Tampones (Química) , Anhidrasas Carbónicas/farmacología , Bovinos , Ciclohexilaminas , Etanolamina , Datos de Secuencia Molecular , Miosinas/farmacología , Ovalbúmina/farmacología , Alineación de Secuencia , Albúmina Sérica Bovina/farmacología , Suspensiones , TrometaminaRESUMEN
The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544-546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066-1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti-P. aeruginosa antibody but was superior to the recombinant anti-P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.