High-cell-density fermentation studies of a recombinantE. coli that expresses atrial natriuretic factor

Summary Studies are presented on the fermentation of recombinantEscherichia coli that express rat atrial natriuretic factor (ANF) as a fusion protein. Our objective was to achieve high cell density while maintaining ANF expression at the same level as observed in shake flasks. Improved fermentation conditions included: maintaining glucose concentrations at 1 g/l, using an enriched medium, adding concentrates of medium throughout the fermentation, and blending oxygen for adequate aeration. Cell densities of 12 g/l (dry weight) were achieved, which represented a 10-fold increase over non-improved conditions, while maintaining ANF levels at 7 mg/g of dry cell mass. When galactose was used as an initial carbon source or as a feed supplement, there was a 2-3-fold increase in the expression of ANF from these high-cell-density fermentations. The recombinant ANF was biologically active.     

Regulated production of recombinant echistatin by yeast

Summary The -mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.     

Antigenic subunit of the polypeptide antigenic complex of the Melvin strain of Neisseria gonorrhoeae.

An antigenic subunit of molecular weight 66,000 daltons has been isolated from the antigenic complex of the Melvin strain of $\text{Neisseria gonorrhoeae}$. Incubation of the complex in 8M urea at room temperature for four hours resulted in the dissociation of the subunit from the complex. It was separated from the complex by chromatography of the incubation mixture on a Sepharose 6B column in 50 mM ammonium bicarbonate pH 8.5 without 8M urea and further purified by affinity chromatography. This communication reports on a newly isolated antigenic protein devoid of LPS present in the bacteria.     

Purification and characterization of a functionally homogeneous 60-kDa species of the retinoblastoma gene product.

The retinoblastoma susceptibility gene (RB) encodes a 928-amino acid protein (pRB) that is hypothesized to function in a pathway that restricts cell proliferation. The immortalizing proteins from three distinct DNA tumor viruses (SV40 large T antigen, adenovirus E1a, and human papilloma virus Type 16 E7) have been shown to interact with RB protein through two noncontiguous regions comprised of amino acids 393-572 (domain A) and 646-772 (domain B). We constructed a truncated form of RB (RB p60) that retains these two domains but eliminates the N-terminal 386 amino acids of RB. RB p60 was expressed in Escherichia coli in inclusion bodies. After solubilization, it was refolded in the presence of magnesium chloride, and the active protein was isolated with an E7 peptide affinity column. The protein that elutes from this column is functionally homogenous in its ability to bind immobilized E7 protein. Thermal denaturation studies provide additional evidence for the conformational homogeneity of the isolated protein. This purification scheme allows the isolation of significant amounts of RB p60 protein that is suitable for structural and functional studies.     

Production of hepatitis B surface antigen (preS2 + S) by high-cell density cultivations of a recombinant yeast.

A high-cell density bioprocess has been developed for the production of hepatitis B surface protein (preS2 + S) by recombinant yeast. This fed-batch process utilizes a growth medium containing yeast extract, soy peptone and glucose which was fed at a constant rate to maintain cells in a respiratory state. Cell densities of up to 60 g l-1 dry weight were achieved, which represented a 6-fold increase over those from batch bioprocesses. This increase in cell mass was attained without compromising specific activity; therefore, volumetric productivities of six times those of batch bioprocesses were achieved.     

Production of cytotoxic proteins in Escherichia coli: a fermentation process for producing enzymatically active HIV-1 protease

Summary Two fermentation processes for the tryptophan-regulated expression of active HIV protease (HIV-1 prt) in Escherichia coli are described. Since overexpression of HIV-1 prt results in cell death, stringent control of product expression was necessary to attain high enzyme levels. Such control was achieved by separation of growth and production phases in a two-step process or by implementation of nutrient feed in a one-step process. When the two-stage process was used, soluble product was detectable only when induction occurred at low culture density (A ₅₅₀ < 3.5). Short induction periods of 1–2 h and rapid harvesting were necessary to recover active product. Similar results were obtained when the single-stage process was operated at 37°C; however, cultivation and induction at 28°C resulted in active enzyme formation following induction at increased cell density (A ₅₅₀=10). Offprint requests to: W. K. Herber     

Galactose-regulated expression of hepatitis B surface antigen by a recombinant yeast

Summary A yeast expressing the hepatitis B surface antigen (HBsAg) gene under the control of a regulated promoter was grown in batch and fed-batch modes. In batch fermentations, the addition of galactose caused the rapid production of HBsAg. Cells grown in fed-batch mode did not produce HBsAg unless the conditioned medium was replaced prior to induction.     

Physiological effects of TGF(alpha)-PE40 expression in recombinant Escherichia coli JM109

Physiological effects of isopropyl-thiogalactopyranoside (IPTG) induction were examined in Escherichia coli strain JM109 expressing a fusion protein composed of transforming growth factor alpha and a 40-kD portion of Pseudomonas aeruginosa exotoxin A (TGF(alpha)-PE40) under control of the tac promoter. Fermentations at the 15-L scale in complex medium compared growth and metabolite profiles of the untransformed JM109 host strain, the strain transformed with the vector lacking the TGF(alpha)-PE40 open reading frame (JM109[pKK2.7]), and the strain with the complete plasmid for TGF(alpha)-PE40 expression (JM109[pTAC-TGF57-PE40]). Metabolite and growth profiles of JM109 (pTAC-TGF57-PE40) cultures changed significantly in IPTG-induced versus uninduced cultures. Prior to induction, glucose was metabolized to acetate or completely oxidized to CO(2). Following induction, pyruvate was also excreted in addition to acetate. In the absence of inducer, pyruvate was excreted by JM109 (pTAC-TGF57-PE40) only when dissolved oxygen levels fell to less than 10% of saturation (microaerobic rather than anaerobic conditions). The untransformed JM109 host strain or JM109 (pKK2.7) did not excrete pyruvate in the presence or absence of inducer, although JM109 (pKK2.7) exhibited a pattern of growth following addition of IPTG that closely resembled JM109 (pTAC-TFG57-PE40). Fermentations of JM109 (pTAC-TFG57-PE40) in a synthetic medium supported lower expression levels, but resulted in similar alterations in metabolite profiles. Induction in synthetic medium resulted in pyruvate excretion without further acetate accumulation. Taken together, these data suggest that one consequence of TGF(alpha)-PE40 expression in JM109 is altered patterns of pyruvate oxidation. (c) 1992 John Wiley & Sons, Inc.     

Harvesting recombinant microbial cells using crossflow filtration.

A contained, crossflow filtration (CFF) membrane system is described for harvesting Saccharomyces cerevisiae and Escherichia coli cells. This system is portable and can be cleaned and sanitized in place. Low- and high-cell density (LCD, HCD) fermentations of recombinant cells in 10- to 200-l volumes were used as the starting material. LCD fermentations, up to 8.3 g l-1 dry weight (dcw) of S. cerevisiae, with volumes of 10 to 200 l were harvested and diafiltered in 0.5 and 1.5 h, respectively. HCD 200-l fermentations of S. cerevisiae (47-63 g l-1 dcw) were harvested and diafiltered in approximately 2 h. E. coli fermentations, LCD and HCD (up to 16.2 g l-1 dcw), of 200-l volumes were harvested and diafiltered in 2.3 h while employing 14 and 75 ft2 of membrane area, respectively. Using hollow fiber or flat sheet membranes from different sources, cell harvesting times were less than 2.5 h. These studies demonstrate that CFF is an efficient method for harvesting and diafiltering recombinant S. cerevisiae and E. coli cells from fermentation broth.