Wayne K. Herber

The structure of human acidic fibroblast growth factor and its interaction with heparin

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.

Determination of carbon sources in fermentation media using high-performance anion-exchange liquid chromatography and pulsed amperometric detection

Abstract The technique of high pH anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) has been adapted for the rapid determination of common carbohydrates present in fermentation broths. Simple water dilution of filtered fermentation broth samples was the sole sample preparation step required. The samples were analyzed using a Dionex CarboPac PA1 column with 150 m M NaOH as the mobile phase at a flow-rate of 1 ml/min and a total run time of 20 min. A gradient method was also developed to resolve species which exhibited similar t R in the isocratic procedure. Among the analytes examined in this study were ethanol, glycerol, galactose, glucose, mannose, fructose, raffinose, ribose and lactose. Examples from several microbial fermentations using chemically defined or complex medium are presented.

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

SummaryTwo 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 (A550 < 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 (A550=10).

Nanogram quantitation of secreted protein in a recombinant yeast fermentation using an immuno-ligand assay

A liquid-phase immuno-ligand assay has been developed for quantitative determination of recombinant tick anticoagulant protein (rTAP) secreted in yeast fermentations. A polyclonal anti-TAP antibody was labeled with biotin or fluorescein. Labelled antibodies were used in a non-competitive sandwich format to capture rTAP from solution, then reacted with urease-conjugated anti-fluorescein antibody. Detection of the immune complex was by a commercially available silicon-based potentiometric sensor which measures urease activity. Sample throughput was 90 samples per 7 h with a 2 h incubation time. The range of the standard curve was 0.1-10 ng/ml with an assay sensitivity of 0.025 ng/ml. For a mid-range concentration of 1 ng/ml, intraday and interday method precision was determined to be 1.031 +/- 0.061 and 1.077 +/- 0.026 ng/ml, respectively. Typically, spiked samples of 1 microgram rTAP/ml fermentation medium required dilutions of 1/1000 to generate a response in the mid-range of the standard curve. This assay provides a convenient method to quantitate product expression in multiple fermentation samples within 3 h after sampling. In addition, a modified version of the assay was developed which provided accurate results within 1 h of sample acquisition.