J. Microbiol. Biotechnol. (2011), 21(10), 1053–1056 doi: 10.4014/jmb.1105.05014 First published online 28 July 2011 Production of a Platelet Aggregation Inhibitor, Salmosin, by High Cell Density Fermentation of Recombinant Escherichia coli Seo, Myung-Ji 1 , Hak-Jong Choi 2 , Kwang-Hoe Chung 3 , and Yu-Ryang Pyun 4 * Department of Chemistry, Box H, Brown University, Providence, RI 02912-9108, USA Department of Microbiology and Immunology, Northwestern University, Chicago, IL 60611, USA Deaprtment of Applied Bioscience, CHA University, Sungnam 463-836, Korea Department of Biotechnology, Yonsei University, Seoul 120-749, Korea Received: May 12, 2011 / Revised: June 22, 2011 / Accepted: July 12, 2011 Optimal conditions for a high cell density fermentation were investigated in a recombinant Escherichia coli producing salmosin, a platelet aggregation inhibitor. The optimized carbon and nitrogen sources were glycerol 10 g/l, yeast extract 30 g/l, and bacto-tryptone 10 g/l, yielding the dry cell weight (DCW) of 10.61 g/l in a 500 ml flask culture. The late-stage induction with 1% L-arabinose in a 5 l jar fermentor showed the highest DCW of 65.70 g/l after 27 h of the fed-batch fermentation. Around 2,200 mg/l of the protein was expressed as an inclusion body that was then refolded to obtain the active salmosin of 96 mg/l. We also confirmed the inhibitory activity against platelet aggregation of the active salmosin from the high cell density fermentation. Keywords: High cell density fermentation, platelet aggregation inhibitor, recombinant Escherichia coli, salmosin Disintegrin is a cysteine-rich low-molecular-weight polypeptide containing the Arg-Gly-Asp (RGD) sequence, which is recognized by various integrins. Disintegrin has a function of inhibiting the aggregation of fibrinogen-dependent platelets by selectively binding to integrins GP IIb-IIIa, which are expressed in platelets [4, 6, 21]. To date, several disintegrins have been isolated from snake venoms and their inhibitory activities against platelet aggregation proved; applaggin from Agkistrodon piscivirous [21], halysin from Agkistrodon halys [9], and saxatilin from Gloydius saxatilis [8]. As another disintegrin, salmosin composed of 73 amino acids was isolated from Agkistrodon halys brevicaudus [11] and its functions demonstrated to inhibit platelet aggregation and tumor angiogenesis, and suppress metastatic tumor growth as well as induce apoptosis by disorganizing focal adhesions [7]. High cell density fermentation is a useful process for improving the production yield of valuable recombinant proteins. In the high cell density fermentation, however, there are several problems including nutrient depletion, exhaustion of dissolved oxygen, and by-products formation causing the inhibition of cell growth and decrease of the desired proteins. To address these problems, the fed-batch fermentation has been applied together with the developments of nutrient feeding strategies including constant feeding, exponential feeding, and indirect feeding such as pH-stat and DO-stat [18]. Escherichia coli has proven to be an appropriate host for recombinant protein expression and production because of the relatively easy constructions of foreign proteins, simple cultivation for their productions using inexpensive medium, and the easy scale-up by a short fermentation cycle [10]. However, the heterologous expressions of foreign eukaryotic proteins in E. coli often lead to the formation of inclusion bodies, by which the number of downstream processes such as solubilization, refolding, and several purification steps could be needed, causing the loss of production yields [3]. Despite these bottlenecks, the recombinant E. coli system has been widely used for the mass productions of foreign proteins with a high cell density. The high cell density fermentation of recombinant E. coli with the inducible promoter system is usually achieved with two separate phases [19]. The cells are grown to a high cell density under the optimized growth conditions such as culture medium, pH, and temperature in the growth phase, where the expression of target protein is kept at a *Corresponding author Phone: +82-2-312-5108; Fax: +82-2-312-5172; E-mail: yrpyun@yonsei.ac.kr # Supplementary data for this paper are available on-line only at http://jmb.or.kr.