RESUMEN
BACKGROUND: Auto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level. OBJECTIVES: In this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium. METHODS: First, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression). RESULTS: Auto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture. CONCLUSION: The medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.
Asunto(s)
Medios de Cultivo/química , Escherichia coli/genética , Glucosa/metabolismo , Lactosa/metabolismo , Proteínas Recombinantes/genética , Angiotensinógeno/genética , Catalasa/genética , Técnicas de Cultivo de Célula , Expresión Génica/efectos de los fármacos , Glucosa/química , Operón Lac , Lactosa/química , Regiones Promotoras Genéticas , Proteínas Recombinantes/metabolismoRESUMEN
Renin is the rate limiting enzyme in the renin-angiotensin (RA) system that regulates blood pressure and electrolyte balance. In this study, we investigated the renin inhibitory effect of a royal jelly (RJ)-derived peptide. A dipeptide YY was isolated from the digested fraction of RJ proteins by proteases and was found to inhibit human renin activity. The inhibition constant (Ki) of YY was estimated to be 10 microM when the Km was 0.16 microM using sheep angiotensinogen as the substrate. The peptide was observed to lower blood pressure in spontaneously hypertensive rats.
Asunto(s)
Dipéptidos/farmacología , Ácidos Grasos/química , Proteínas de Insectos/química , Renina/metabolismo , Angiotensinógeno/genética , Angiotensinógeno/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Células CHO , Cromatografía Líquida de Alta Presión , Cricetinae , Cricetulus , ADN Complementario/genética , Dipéptidos/química , Dipéptidos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos/metabolismo , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Hipertensión/fisiopatología , Hipertensión/prevención & control , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/metabolismo , Estructura Molecular , Péptido Hidrolasas/metabolismo , Ratas , Ratas Endogámicas SHR , Proteínas Recombinantes/metabolismo , Renina/genética , Sistema Renina-Angiotensina/efectos de los fármacos , OvinosRESUMEN
The effect of laughter therapy on the plasma levels of renin, angiotensinogen, and prorenin was investigated in patients with type 2 diabetes. In the diabetic patients, the mean plasma renin concentrations were 24.6+/-12.1 ng/ml/h in the first observation (at the beginning of laughter therapy), 8.2+/-3.4 ng/ml/h in the second observation (three months after the beginning of laughter therapy) and 7.7+/-1.7 ng/ml/h in the third observation (six months after the beginning of laughter therapy). The mean plasma angiotensinogen concentrations in the 1st, 2nd and 3rd observations were 0.19+/-0.08, 0.47+/-0.12, 0.42+/-0.14 microg/ml, respectively. The mean plasma prorenin concentrations in the 1st, 2nd and 3rd observations during the laughter therapy were 195.1+/-66.2, 193.4+/-88.2 and 170.7+/-52.5 pg/ml, respectively. Plasma renin concentrations were significantly decreased (p<0.05) by the therapy. Subnormal concentrations of plasma angiotensinogen were found in the 1st observation and increased significantly (p<0.05) to the normal range after the therapy. Plasma prorenin concentration only slightly changed during the laughter therapy. Other biochemical parameters remained unchanged during the laughter therapy. These results indicated that a long-term laughter therapy changed the plasma components of renin-angiotensin system in patients with diabetes. Thus, laughter therapy can be used as non-pharmacological treatment for the prevention of diabetic microvascular complications.