Journal of Biotechnology 120 (2005) 11–24 Modelling of translation of human protein disulfide isomerase in Escherichia coli—A case study of gene optimisation Olli Niemitalo a,1 , Antje Neubauer b,d,1 , Ulf Liebal a , Johanna Myllyharju b,d , Andr´ e H. Juffer c,d , Peter Neubauer a,d,∗ a Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, Oulu, Finland b Collagen Research Unit, Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland c Triacle Biocomputing, Oulu, Finland d Biocenter Oulu, Oulu, Finland Received 30 December 2004; received in revised form 21 April 2005; accepted 4 May 2005 Abstract Recombinant human protein disulfide isomerase (PDI) was expressed in vivo in Escherichia coli using a non-optimised gene sequence and an optimised sequence with four 5 ′ codons substituted by synonymous codons that take less time to translate. The optimisation resulted in a 2-fold increase of total PDI concentration and by successive optimisation with expression at low temperature in a 10-fold increase of the amount of soluble PDI in comparison with the original wild-type construct. The improvement can be due to a faster clearing of the ribosome binding site on the mRNA, elevating the translation initiation rate and resulting in higher ribosome loading and better ribosome protection of the PDI mRNA against endonucleolytic cleavage by RNase. This hypothesis was supported by a novel computer simulation model of E. coli translational ribosome traffic based upon the stochastic Gillespie algorithm. The study indicates the applicability of such models in optimisation of recombinant protein sequences. © 2005 Elsevier B.V. All rights reserved. Keywords: Codon usage; Gillespie algorithm; Translation; PDI; Recombinant protein; Escherichia coli; Computer simulation ∗ Corresponding author. Tel.: +358 8 553 2384; fax: +358 8 553 2304. E-mail address: peter.neubauer@oulu.fi (P. Neubauer). 1 Authors contributed equally to the results. 1. Introduction The genetic code is degenerate in that a number of synonymous codons code for the same amino acid. This has promoted the evolution of codon usage biases, i.e. the favouring of the use of some codons over oth- 0168-1656/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2005.05.028