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Download Ingeniería de proteínas en una celulasa de hongos
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Universidad de Chile Facultad de Ciencias Físicas y Matemáticas Departamento de Ingeniería Química y Biotecnología Presenta: Marcela Vega Profesora Guía: Oriana Salazar p y Expression systems Are easy to work, manipulate and cheaper than other cells ▪ Bacterias, don’t Bacterias, don t make postraductionals modifications ▪ E. coli ▪ Levaduras ▪ S. cerevisiae, makes hyperglycosylation ▪ P. pastoris, is good secreting proteins General objetive Complete, clone and express an endoglucanase from fungus Specific objetives Get the complete sequence of the target enzyme Clone the gene into a expression plasmid Evaluate the gene expression in the expression system of E. coli, S. cerevisiae and P. pastoris Determine and compare the production and specific activity of the enzyme produced by the three diferent expression system E Enzyme expression i Activity y studies in native and recombinant systems PCR to get the complete sequence Kinetics of the enzyme production Clone the enzyme into a expression plasmid Purification of the cultures Recombinant expression Compare the activity and production of the enzyme Study of the protein expression and the enzymatic activity of Ganoderma applanatum in a liquid culture Electrophoresis [kda] 8° 10° 12° 14° 16° 19° 21° 12° 14° 16° 19° 21° 118‐ 90‐ 50‐ 34‐ 26‐ 19‐ Zymograms [kda] 118‐ 90‐ 90 50‐ 8° 10° Identified sequence so far bp 1500‐ 1000‐ 800‐ 600‐ 500‐ 400‐ 300‐ 200‐ M Producto PCR Missing fragment bp 1500‐ 1000‐ 800‐ 6 600‐ 500‐ 400‐ 300‐ 200‐ M Producto PCR Missing fragment Complete sequence Secuencie complete bp 1500‐ 1000‐ 600‐ 600 500‐ 400‐ 300‐ 200‐ M Producto PCR Sequences bp 1500‐ 1000‐ 1000 800‐ 600‐ 500‐ 400‐ 300‐ 200‐ M Producto PCR bp 1500‐ 1000 1000‐ 800‐ 600‐ 500‐ 400‐ 300‐ 200‐ M Producto PCR bp 1500‐ 1000‐ 600‐ 500‐ 400‐ 300‐ 200‐ M Producto PCR Sequences bp 1500‐ 1000‐ 1000 800‐ 600‐ 500‐ 400‐ 300‐ 200‐ M bp M Recombinant expression 1500‐ bp 1500‐ 800‐ 600‐ 500‐ 400‐ 1000 1000‐ 800‐ 600‐ 500‐ 400‐ 300‐ 300‐ 200‐ 200‐ 1000 1000‐ Analize the sequencing of the obteined fragment Get the complete sequence Clone the gene sequence into a expression vector Express the enzime on the three systems named Make the kinetic studies of the enzymatic production and specific activity
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