Production of recombinant HIV‐1 Nef (negative factor) protein using Pichia pastoris and a low‐temperature fed‐batch strategy

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.

Continuous Cultivation as a Tool Toward the Rational Bioprocess Development With Pichia Pastoris Cell Factory

“…On the contrary, bioreaction rates such as intracellular reactions, proteolysis and cell growth diminish. Reduced proteolysis at low temperatures is the result of decreased protease activity rather than decreased proteases production (Sirén et al, 2006 ). Thus, some studies have focused mainly on this cultivation parameter.…”

Comparative genome‐scale analysis of Pichia pastoris variants informs selection of an optimal base strain

“…Despite the advantages of this host, the interchangeable use of various base strains of P. pastoris available today has led to reports of substantially variable performance. Productivity (Goncalves, 2013;Hochstrasser, Lüscher, De Virgilio, Boller, & Wiemken, 1998;Seo, & Rhee, 2004), growth (Hochstrasser et al, 1998;Seo, & Rhee, 2004;Sirén et al, 2006), proteolytic activity (Salamin, Sriranganadane, Léchenne, Jousson, & Monod, 2010;Sinha, Plantz, Inan, & Meagher, 2005;Sirén et al, 2006), and glycosylation (Blanchard et al, 2008;Tzeng & Jiang, 2004) have varied substantially among strains depending on the product expressed. Such inconsistencies underscore the need for a systematic evaluation of host properties to inform the choice of a standard base strain.…”

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

“…They grow fast to high cell densities, even in chemically defined media. S. cerevisiae has a long history of use in industrial fermentation, but P. pastoris often achieves higher expression levels and can use methanol as a sole carbon source (Sirén et al, 2006), which however, is not always desired due to high oxygen consumption and its consequences (Zavec et al, 2020). The lack of suitable chaperone proteins may lead to improper folding, and the glycosylation patterns are often unacceptable for the production of mammalian proteins in yeast.…”