Short communication Over-production of human interferon-g by HCDC of recombinant Escherichia coli V. Babaeipour, S.A. Shojaosadati * , S.M. Robatjazi, R. Khalilzadeh, N. Maghsoudi Biotechnology Group, Chemical Engineering Department, Faculty of Engineering, Tarbiat Modarres University, P.O. 14155-143, Tehran, Iran Received 19 February 2006; received in revised form 24 May 2006; accepted 7 July 2006 Abstract A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-g). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h 1 at the beginning of feeding and then it was reduced to 0.4 h 1 at induction time. The final concentration of biomass and IFN-g was reached to 115 g l 1 (DCW) and 42.5 g(hIFN-g)l 1 after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-g (rhIFN-g) were obtained 0.37 g(hIFN-g)g 1 DCW and 2.57 g(hIFN-g)l 1 h 1 , respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet. # 2006 Elsevier Ltd. All rights reserved. Keywords: Human interferon-g; Fed-batch; Escherichia coli; Specific growth rate; Feeding strategy; High cell density cultivation (HCDC) 1. Introduction HIFN-g is a glycosylated protein with a total molecular size of 25 kDa and composed of 143 amino acid residues [1,2]. However recombinant hIFN-g (rhIFN-g) expressed in E. coli is not glycosylated and has a molecular weight of 17 kDa, but is still active physiologically [3]. E. coli is one of the most widely used hosts for the production of heterologous proteins [4]. Because most proteins are accumulated intracellularly in recombinant E. coli, productivity is proportional to the final cell density and the specific productivity (i.e. the amount of product formed per unit time). The high cell density cultivation (HCDC) techniques for cultivation of E. coli have been developed to improve productivity. Fed-batch processes have most often been used to obtain high cell densities [5]. In the fed-batch system, feeding strategy is critical to the success of HCDC, because it affects the metabolic pathway fluxes, and consequently affects the maximum attainable cell concentration, the specific productivity of recombinant proteins and formation of by-products [5–7]. Various nutrient feeding strategies have been employed for the HCDC of E. coli [5]. Simple feeding strategies such as: constant feeding rate, variable feeding rate, and exponential feeding rate have been successfully used to obtain HCDC of E. coli [5,6]. The exponential feeding method in fed-batch cultivation is probably one of the most widely used methods for growing cells to high cell densities, since in this methodology it is easy to implement the process and manipulate of specific growth rate [6,8–10]. It has been shown that the specific productivity of recombinant proteins obtained by HCDC is generally lower than that obtained by batch culture [6,11]. A possible reason is that many fed-batch processes are run at low specific growth rates and under substrate limited conditions before and after induction and consequently most available carbon and energy sources have to be used for maintenance requirements [11]. On the other hand, the higher specific growth rate during production [12] or even before induction [13,14] can increase recombinant protein production, presumably via higher ribosome content [14]. Also achieving substrate non-limited conditions can reduce stress responses before and after induction [11,14,15]. Therefore, manipulating specific growth rate, as a key process parameter that represents the dynamic behavior of microorganisms during fermentation, at an www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 112–117 * Corresponding author. Tel.: +98 21 88005040; fax: +98 21 88005040. E-mail address: shoja_sa@modares.ac.ir (S.A. Shojaosadati). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2006.07.009