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Production of recombinant HIV‐1 Nef (negative factor) protein using Pichia pastoris and a low‐temperature fed‐batch strategy
Author(s) -
Sirén Noora,
Weegar Jan,
Dahlbacka John,
Kalkkinen Nisse,
Fagervik Kaj,
Leisola Matti,
Von Weymarn Niklas
Publication year - 2006
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060001
Subject(s) - pichia pastoris , proteases , yeast , recombinant dna , pichia , chemistry , protease , chromatography , plasmid , biochemistry , biology , gene , enzyme
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 pPICZαA 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.