RESUMEN
Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colorantes Fluorescentes , Proteínas de la Membrana/biosíntesis , Ácido Nitrilotriacético , Receptor de Adenosina A2A/metabolismo , Transferasas/metabolismo , Proteínas Bacterianas/genética , Cromatografía en Gel , Proteínas Fluorescentes Verdes , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Receptor de Adenosina A2A/genética , Proteínas Recombinantes de Fusión/química , Sensibilidad y Especificidad , Transferasas/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)Asunto(s)
Hongos/fisiología , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiología , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Fusarium/fisiología , Espectroscopía de Resonancia Magnética , Phytophthora/fisiología , Enfermedades de las Plantas , Raíces de PlantasRESUMEN
The primary immune response to Helix pamatia haemocyanin (HPH) was investigated in sixty-one patients with various clinical signs of malignant melanoma and compared to that of controls. Anti-HPH antibody response was only found abnormal in patients with widespread disease and visceral metastases, apparent from decreased 7S and increased 19S antibody levels. In vitro HPH-induced lymphocyte transformation, in contrast, was not only decreased in this late stage, but also in part of the patients in the beginning of the disease and was then correlated with subsequent tumour recurrence within 6 months. Generally, patients with positive lymphocyte transformation showed significantly higher 7S anti-HPH titres (mean 3-3 +/- 2-4 than patients without (1-7 +/- 1-6). In contrast to the lymphocyte transformation reaction, anti-HPH antibody response was not correlated with subsequent tumour recurrence. The same phenomenon of decreased 7S and increased 19S antibody response as in the melanoma patients with far advanced disease was observed to a less extent in controls with ageing. Anamnestic antibody responses to diphtheria and tetanus toxoid and serum IgG and IgA levels were found normal. Elevated serum IgM levels were more frequently observed in patients with localized (19/31) and disseminated disease (11/29) than in controls (2/31). These results suggest that melanoma patients develop impaired T-lymphocyte functions in final stages with visceral metastases. A previous study showed that minor defects in lymphocyte function in an early stage as measured by HPH-induced lymphocyte transformation have predictive value for subsequent tumour recurrence.