High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris

Abstract

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

“…AOX1-deleted transformants could grow slowly in the methanol medium because they had another alcohol oxidase encoded by the AOX2 gene, which is expressed more weakly than the AOX1 gene . The frequency of AOX1 disruption by double crossing-over (37%) was identical to the frequency reported for the production of hepatitis B antigen by Cregg et al (1987), tumour necrosis factor by Sreekrishna et al (1989) and human single-chain urokinase-type plasminogen activator (Tsujikawa et al, 1996).…”

Physiological and technological aspects of large-scale heterologous-protein production with yeasts

“…Multiple-integration systems have also been developed for the methylotrophic yeasts Pichia pastoris (Sreekrishna et al 1989) and Hansenula polymorpha (Roggenkamp et al 1986). These systems do not rely on targeting of the expression vector to reiterated chromosomal sequences.…”

Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement

“…For Mut s strains, due to their slow utilization of methanol, a mixed feed of glycerol and methanol is commonly employed in the fermentation induction phase; glycerol functions as an efficient substrate for cell growth and target protein production while methanol functions as an inducer. With this strategy, various proteins have been successfully expressed in either fed-batch or continuous operation mode by Mut s strains [1,2,14,19]. In all of these studies, the optimization of mixed feeding strategy was based on arbitrary ratios of the two substrates in the feed solution.…”