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  • Title: Production of recombinant HIV-1 Nef (negative factor) protein using Pichia pastoris and a low-temperature fed-batch strategy.
    Author: Sirén N, Weegar J, Dahlbacka J, Kalkkinen N, Fagervik K, Leisola M, von Weymarn N.
    Journal: Biotechnol Appl Biochem; 2006 Jun; 44(Pt 3):151-8. PubMed ID: 16551271.
    Abstract:
    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.
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