Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy.
In the present paper we describe the cloning and extracellular expression of the HIV-1 Nef (negative factor) protein utilizing the yeast Pichia pastoris, as well as the successful use of a low-temperature fed-batch strategy for decreasing end-product degradation by proteases. The nef gene in a pPICZalphaA vector was integrated into the genome of three different P. pastoris strains, namely X-33, GS115 and KM71H. On the basis of its efficient growth and production characteristics the wild-type strain (X-33) was found to be the best choice. The decreased end-product degradation at low temperatures was not due to lower amounts of proteases but due to their diminished activity. The yield of biomass from methanol was improved 1.44-fold utilizing the low-temperature strategy compared with the standard fermentation. Purification of histidine-tagged Nef was performed in one step using a Ni(2+)-nitrilotriacetate-Sepharose column. The purified product was characterized by SDS/PAGE, Western blotting, matrix-assisted laser-desorption ionization-time-of-flight MS, reversed-phase HPLC and N-terminal-sequence analysis.
Laboratory of Bioprocess Engineering, Department of Chemical Technology, Helsinki University of Technology, P.O. Box 6100, FI-02015 TKK, Finland. noora.siren@tkk.fi
Noora
N
Sirén
Jan
J
Weegar
John
J
Dahlbacka
Nisse
N
Kalkkinen
Kaj
K
Fagervik
Matti
M
Leisola
Niklas
N
von Weymarn
D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015693:Gene Products, nef / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D013997:Time Factors
D015693:Gene Products, nef; D011994:Recombinant Proteins