Alternative production process strategies in E. coli improving protein quality and downstream yields Jordi Ruiz, Jaume Pinsach, Gregorio A ´ lvaro, Glo ` ria Gonza ´ lez, Carles de Mas, David Resina, Josep Lo ´ pez-Santı ´n * Departament d’Enginyeria Quı´mica, Escola Te `cnica Superior d’Enginyeria, Unitat de Biocata `lisi Aplicada associada al IIQA (CSIC), Universitat Auto `noma de Barcelona, Edifici Q, 08193-Bellaterra, Spain 1. Introduction Nowadays, biotechnology is seen as an important tool for the sustainable industrial development. This requires the develop- ment of new enzymes, processes, products and applications [1]. Enzymes are used in a wide range of applications and industries [2], and they are a promising alternative/complement to chemical synthesis for producing chiral synthons that are used as building blocks to obtain highly active pharmaceuticals. Aldolases catalyze C–C bond formation with defined stereo- chemistry yielding enantiomerically pure products, even when the starting materials are non-chiral substrates [3]. Rhamnulose 1-phosphate aldolase (RhuA) catalyses the reversible cleavage of L-rhamnulose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde in vivo. The potential of RhuA as biocatalyst in asymmetric synthesis is based on the fact that the reaction can be reversed in vitro, and it accepts a wide range of both natural and non-natural substrates for the stereoselective synth- esis of carbohydrates and other derivatives of pharmaceutical interest [4–7]. The production of recombinant proteins in Escherichia coli has been the subject of numerous studies. Several genetic as well as cultivation strategies have been developed to increase recombi- nant protein yields [8–10]. However, limitations still exist to produce them at high levels in a biological active form, and proteolysis has been reported to impact recombinant protein production processes [11–13]. Little work has been done on the control of proteolysis in high cell density fed-batch cultures [14] and although affinity tags have been reported to have a positive impact on yield, solubility and even folding and reduction of proteolysis [15], partial proteolysis has been identified as the cause for reduced IMAC recovery yields [16]. Process Biochemistry 44 (2009) 1039–1045 ARTICLE INFO Article history: Received 30 May 2008 Received in revised form 14 May 2009 Accepted 19 May 2009 Keywords: E. coli Protein quality One step purification-immobilization Substrate in excess fed-batch cultures Continuous inducer dosage Aldolase ABSTRACT Process strategies for production of recombinant rhamnulose 1-phosphate aldolase (RhuA) in Escherichia coli were found to have an important impact on downstream processing when preparing the enzyme for its use as immobilized biocatalyst. First, a continuous inducer feed was implemented in substrate limited fed-batch cultures to overexpress RhuA with a hexa histidine-tag (6xHis-tag) at its N-terminus. The final specific RhuA level was 180 mg g À1 DCW, but the final specific enzyme activity (1.7 AU mg À1 RhuA) was considerably lower than expected. Only 55% of immobilization yield was achieved when immobilized metal affinity chromatography (IMAC) was used to purify and immobilize RhuA from cellular lysate in a single step. Western blot analyses showed that only 20% of overexpressed RhuA kept the whole 6xHis- tag at the end of the culture due to partial proteolysis. Two different growth strategies improved protein quality and immobilization yield: (i) Temperature reduction to 28 8C in substrate limited operation decreased proteolysis and allowed higher specific activities, 210 mg g À1 DCW. The enzyme activity increased to 4 AU mg À1 RhuA and purification-immobilization yield to 93%. (ii) A novel fed-batch operational procedure, working at high glucose concentration was implemented. High aldolase levels, 233 mg g À1 DCW, were reached at the end of the culture. The final enzyme activity was also higher than 4 AU mg À1 RhuA, and 95% of immobilization yield was achieved. For both cases, Western blot analyses showed that 80–100% of overexpressed RhuA kept the whole 6xHis-tag at the end of the culture, confirming that recombinant protein quality had been improved. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: +34 935811806; fax: +34 935812013. E-mail address: josep.lopez@uab.cat (J. Lo ´ pez-Santı ´n). Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.05.007