RESUMEN
We have raised monoclonal antibodies against KLK6 and constructed a 'sandwich' type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 µg/l±1.37) than in controls (3.39 µg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257-965 µg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Fluoroinmunoensayo/métodos , Calicreínas/sangre , Calicreínas/inmunología , Secuencia de Aminoácidos , Sitios de Unión , Técnicas de Visualización de Superficie Celular , Cristalografía por Rayos X , Femenino , Humanos , Calicreínas/química , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunologíaRESUMEN
Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.
Asunto(s)
Calicreínas/genética , Línea Celular , Clonación Molecular/métodos , Humanos , Proteínas Recombinantes , Recombinación GenéticaRESUMEN
Only one transcript for KLK10 was identified by RT-PCR in lung tissue, whereas KLK11 expressed at least four alternative transcripts. Quantitative analysis of KLK10 and KLK11 expression levels was assessed by real-time PCR, in a cohort of 47 patients with non-small-cell lung cancer (NSCLC). Expression levels of these genes were widely distributed in the population studied. Multivariate analysis revealed a correlation between KLK10 over-expression and the squamous cell carcinoma histotype (p=0.034). There was no correlation between gene expression and patient survival. Overall, both genes behaved similarly (p<0.001). These results suggest a co-regulation of KLK10 and KLK11 expression in lung and a lack of KLK10 suppressor role in NSCLC. Finally, our findings indicate that these genes are likely involved in normal physiology processes in bronchus.
