Production of soluble recombinant proteins in Escherichia coli: Effects of process conditions and chaperone co-expression on cell growth and production of xylanase Kamna Jhamb, Debendra K. Sahoo ⇑ CSIR – Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India highlights " Low temperature or reduced metabolism favored soluble protein expression. " Co-expression of molecular chaperones resulted in 33–40% soluble Xyn-B expression. " Soluble expression of Xyn-B was reproducible in a scalable semi-synthetic medium. " High E. coli cell O.D. and growth rate could be due to inclusion body formation. article info Article history: Received 18 May 2012 Received in revised form 6 July 2012 Accepted 7 July 2012 Available online 16 July 2012 Keywords: Escherichia coli Inclusion bodies Soluble expression Molecular chaperones Xylanase abstract In this study, effects of temperature, inducer concentration, time of induction and co-expression of molecular chaperones (GroEL–GroES and DnaKJE), on cell growth and solubilization of model protein, xylanases, were investigated. The yield of soluble xylanases increased with decreasing cultivation tem- perature and inducer level. In addition, co-expression of DnaKJE chaperone resulted in increased soluble xylanases though the time of induction of chaperone and target protein had a bearing on this yield. A combination of chaperone co-expression and partial induction resulted in 40% (in DnaKJE) and 33% (in GroEL–GroES) of total xylanase yield in soluble fraction. However, the conditions for maximum yield of soluble r-XynB and maximum % soluble expression of r-XynB were different. Higher expression of sol- uble xylanases in a scalable semi-synthetic medium showed potential of the process for soluble enzyme production. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction The extensive employment in recombinant protein production and plethora of knowledge available on genetics and physiology of gram-negative Escherichia coli has made it to be called as the ‘‘microbial factory’’. The ease of handling, inexpensive growth requirements and the accumulation of the product to higher levels in the cell cytoplasm are other features of this organism which have aided in making it the most sought after expression host. However, not all genes are expressed in E. coli in a facile manner. There are several limitations associated with the production of non-native/heterologous proteins in the prokaryotic system of E. coli. It poses significant problems in post-translational modifica- tions of proteins, having no capacity to bring about glycosylation or disulfide bond formation. As a result, recombinant polypeptides are found to be sequestered within large refractile aggregates of inactive protein known as inclusion bodies (IBs) (Georgiou and Valax, 1996). Recovery of biologically active products from aggre- gated state is typically accomplished by unfolding with chaotropic agents followed by dilution/dialysis into optimized refolding buf- fers. Optimization of the refolding procedure however, requires time consuming efforts and is not conducive to high product yields (Sorensen and Mortensen, 2005). Thus, maximizing the yields of recombinant proteins in a soluble and active form in vivo becomes an alternative to in vitro folding. From an industry perspective, low product yields and high recovery costs of a protein/enzyme from a microbiological source is not acceptable and hence conditions have to be explored which balance heterologous protein production and host physiology to maximize soluble product yields. A number of approaches for the redirection of proteins from IBs into the soluble fraction are de- scribed in the literature. Modification of cultivation conditions to changing a host cell, or use of fusion partners are some of the 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.07.011 ⇑ Corresponding author. Tel.: +91 172 6665324; fax: +91 172 2690632. E-mail address: debsahoo@imtech.res.in (D.K. Sahoo). Bioresource Technology 123 (2012) 135–143 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech