title: Modelling of translation of human protein disulfide isomerase in Escherichia coli-A case study of gene optimisation.
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.
authors.affiliation: Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering, University of Oulu, Oulu, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Niemitalo
authors.forename: Antje
authors.initials: A
authors.lastname: Neubauer
authors.forename: Ulf
authors.initials: U
authors.lastname: Liebal
authors.forename: Johanna
authors.initials: J
authors.lastname: Myllyharju
authors.forename: André H
authors.initials: AH
authors.lastname: Juffer
authors.forename: Peter
authors.initials: P
authors.lastname: Neubauer
mesh_terms: D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D024861:Genetic Enhancement / Q000379:methods*; D006801:Humans; D008957:Models, Genetic; D014176:Protein Biosynthesis / Q000235:genetics*; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D019704:Protein Disulfide-Isomerases