The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide.

There is a great demand for the improvement of mammalian cell production systems such that they can compete economically with their prokaryotic counterparts. Of a number of parameters that need to be explored to accomplish this we have tested the effects of different signal peptides on the synthesis and secretion of Gaussia princeps luciferase in mammalian cells. A series of plasmids were transfected into CHO cells where the coding region for the marine luciferase was fused to the signal peptide coding regions derived from different sources. Both cell extracts and medium samples were analysed for luciferase activity. When the native Gaussia luciferase signal sequence in the vector was substituted by that from human interleukin-2 or albumin then the amount of active recombinant protein produced was substantially reduced, both in transiently and stably transfected cells. Western blotting showed that enzyme activity and protein levels mirrored one another. The major decrease in luciferase activity was shown not to be a result of decreased mRNA levels, indicating the involvement of a post-transcriptional event. When the coding region of human endostatin was fused to that of the Gaussia luciferase signal peptide then an elevated level of secreted endostatin was observed compared to when that of the albumin signal peptide was used. Stable transfection of HepG2 cells with the different signal peptide constructs gave essentially the same results as seen in CHO cells. The overall results indicate that the choice of signal peptide can be imperative to ensure an optimal synthesis and secretion of a recombinant protein in a mammalian cell culture system.

Vectors encoding seven oikosin signal peptides transfected into CHO cells differ greatly in mediating Gaussia luciferase and human endostatin production although mRNA levels are largely unaffected.

The signal peptide of the luciferase secreted by the marine copepod Gaussia princeps has been shown to promote high-level protein synthesis/secretion of recombinant proteins, being far superior to mammalian counterparts. The main aim of the present study was to investigate the effects of seven selected signal peptides derived from oikosins, house proteins of the marine organism Oikopleura dioica, on synthesis/secretion of recombinant proteins. Vector constructs were made in which the coding regions of two naturally secreted proteins, Gaussia luciferase and human endostatin (hEndostatin), were "seamlessly" fused to the signal peptide coding sequences of interest. CHO cells were transfected with the plasmids and populations of stably transfected cells established. The amounts of reporter proteins in cell extract and medium samples were determined and the results compared to those obtained from cells stably transfected with a reference vector construct. In addition, the amounts of luciferase or hEndostatin encoding mRNAs in the cells were determined and related to the protein levels obtained. The levels of reporter protein produced varied greatly among the seven oikosin signal peptides tested. Whereas the oikosin 1 signal peptide resulted in about 40% production of Gaussia luciferase compared to the reference construct, oikosins 2-7 were extremely ineffective (<1%). mRNA levels were not dramatically affected such that inadequate availability of transcript for translation was not the underlying reason for the observations. The oikosin 1 signal peptide was also the most effective regarding synthesis/secretion of hEndostatin. No secreted product was observed using the oikosin 3 signal peptide. Interestingly, the molecular weight of hEndostatin in cell extracts prepared from cells transfected with oikosin 2 and 3 constructs was higher than that using the oikosin 1 signal peptide. The overall findings indicate that the signal peptide affects the efficiency of protein synthesis and secretion through a mechanism operating at the post-transcriptional level. The results described here provide substantial support to our previous observations which suggested that the choice of the signal peptide is imperative when aiming to achieve optimal synthesis and secretion of a recombinant protein using transfected mammalian cells.