Fermentation process for tetrameric human collagen prolyl 4-hydroxylase in Escherichia coli: Improvement by gene optimisation of the PDI/β subunit and repeated addition of the inducer anhydrotetracycline

The collagen prolyl 4-hydroxylases (C-P4Hs) that reside within the lumen of the endoplasmic reticulum (ER) are the key enzymes in the biosynthesis of collagens. The vertebrate enzymes are α2β2 tetramers consisting of two catalytic α subunits and two β subunits that are identical to protein disulfide isomerase (PDI). Cytoplasmic production of an active human C-P4H has recently been described in the Origami (trxB gor) mutant Escherichia coli using a bicistronic vector with independent control of the α and PDI/β subunit expression by the tetA and T5-lac promoters, respectively, enabling sequential induction (Neubauer, A., Neubauer, P., Myllyharju, J., 2005. High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli. Matrix Biol. 24, 59–68). We show here that the yield of active C-P4H in shake flasks is increased 50-fold by improving the expression level of the PDI/β subunit through gene optimisation. We also found that stable expression of the α subunit mRNA in a fed-batch fermentation process requires repeated additions of anhydrotetracycline. This finding may be of a wider general importance for the use of the tetA promoter in fed-batch cultivations, especially if recombinant proteins are expressed during long production phases. We also show that growth of the E. coli Origami strain to high cell density on a complex medium with consecutive sequential induction is difficult to achieve and that optimisation of similarly complicated systems can greatly benefit from the use of quantitative mRNA analysis for the evaluation of transcriptional bottlenecks. The optimisation approach resulted in a fermentation yield of 143 mg L−1 of active C-P4H, corresponding to approximately 7.5% of the total soluble cell protein.