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.

Expression of human tumor necrosis factor-alpha (TNF) and four different TNF analogs has been studied in Pichia pastoris by utilizing the alcohol oxidase gene promoter. TNF expression level in certain transformants accounted for as much as 36% of the soluble protein. TNF expression was stably maintained during high cell density fermentation (100 g dry cell weight/liter) resulting in a TNF production level of 6-10 g/liter. TNF contained in P. pastoris cell lysates was biologically active as determined by its cytotoxic effect on murine L-929 fibroblast cells and the bioactivity was retained for at least 6 months in the lysates stored frozen at -20 degrees C in the presence of protease inhibitor PMSF. TNF expressed in P. pastoris was recognized by monoclonal antibodies prepared against recombinant Escherichia coli derived TNF. TNF purified from P. pastoris has the expected N-terminal amino acid sequence and specific activity of 10(7) units/mg protein. TNF analogs were also expressed at levels comparable to that of native TNF. Three of the four analogs were insoluble when produced in P. pastoris.

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