A stock culture of the strain Streptomyces sp. 2754 was maintained on yeast and malt extract with glucose International Streptomyces Projects Medium 2 agar slant consisting of 0.4% yeast extract (Beijing Aoboxing Biotechnology, Beijing, China), 1.0% malt extract (Beijing Aoboxing Biotechnology), 0.4% glucose and 1.2% agar (pH 7.1). The stock culture was inoculated into 250-ml Erlenmeyer flasks containing 50 ml of medium consisting of 0.5% yeast extract (Beijing Aoboxing Biotechnology), 0.5% glucose, 0.5% tryptone, 0.5% beef extract, 0.4% corn steep liquor (North China Pharmaceutical Corporation, Hebei, China), 1.0% soybean extract (Beijing Comwin Pharm-Culture, Haidian, Beijing, China), 2.0% starch and 0.4% CaCO3 (pH 7.1). The flask culture was incubated on a rotary shaker (250 r.p.m.) at 28 °C for 48 h. The seed culture (5 ml) was transferred into each of the 100, 500-ml Erlenmeyer flasks containing 100 ml of the same medium. The fermentation was carried out at 28 °C for 96 h on a rotary shaker (250 r.p.m.).

The fermentation broth (10 l) was filtered and the filtrate was absorbed on a column of Amberlite XAD-5 (Amberlite XAD-5 Macroporous Resin, Sigma, St Louis, MO, USA) (1 l), after washing with 4 l of water, 3 l of 30% aqueous acetone and 3 l of 50% aqueous acetone, successively, the absorbed materials were eluted with 3 l of 80% aqueous acetone. The fraction eluted with 80% aqueous acetone showed the VEGFR-2 antagonistic activity. The active fraction was concentrated in vacuo and lyophilized to obtain a crude powder (2.8 g). It was then chromatographed on a column of Sephadex LH-20 (Pharmacia, Sweden, 1.5 × 100 cm), developed with methanol. The first yellow fraction showing the activity was concentrated to give a yellow material (12 mg). This material containing enriched 1 was finally purified by medium-pressure liquid chromatography (Yamazen FMI-C pump, Yamazen, Japan; column: Ultra-pack ODS, 15 × 300 mm, 30 per 50 μm; mobile phase: 30% aqueous acetonitrile; flow rate: 1 ml min−1) to yield 1 (1.5 mg) and 1 of 14 mg was obtained from 100 l of the fermentation broth.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account
Continue with Google
Continue with ORCiD