High-yield secretion of multiple client proteins in Aspergillus.
Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50-100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA.
Fernando
F
Segato
André R L
AR
Damásio
Thiago A
TA
Gonçalves
Rosymar C
RC
de Lucas
Fabio M
FM
Squina
Stephen R
SR
Decker
Rolf A
RA
Prade
D001230:Aspergillus / Q000235:genetics* / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005819:Genetic Markers; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D017931:DNA Primers; D005819:Genetic Markers; D011994:Recombinant Proteins