In this series, we are highlighting some of our founder, Dr. Yelena Sheptovitsky’s, favorite manuscripts, publications, and news pieces all relating to the world of protein expression, life science, and CRO’s.
Today we look at the author manuscript from “A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species” by Carolina Attallah*, Marina Etcheverrigaray, Ricardo Kratje, Marcos Oggero
Contents lists available at PubMed
Protein Expression and Purification 132 (2017) 27-33
ABSTRACT
Signal peptides (SPs) are key elements in the production of recombinant proteins; however, little in- formation is available concerning different SP in mammalian cells other than CHO. In order to study the efficiency of different SPs to direct the traffic along the secretory pathway of the green fluorescence protein (GFP) and a scFv-Fc fusion protein; CHO-K1, HEK293 and NS0 cell lines were transfected in a transient and stable way. SP of human azurocidin (AZ), modified human albumin (mSA), modified Cri- cetulus griseus Ig kappa chain V III region MOPC 63 like (mIgk C) and modified human Ig kappa chain V III region VG (mIgk H) were evaluated. The efficiency of SPs to translocate a propeptide across the ER membrane was evaluated by fluorescence microscopy and flow cytometry for the GFP inside the secretory pathway, and by antigen-specific indirect ELISA for the scFv-Fc outside the cell. The mSA SP was successful in directing the secretion of the active proteins in these different types of mammalian cells, regardless of the transgene copy number. The goal of this work was to demonstrate that a modified version of SA SP might be used in different mammalian cells employing the same expression vector.
One area that stands to be mentioned:
Results and discussion
3.1. Evaluation of SP efficiency to target propeptides to the ER by transient transfection
The efficiency of a SP is determined by its ability to target a propeptide to the ER [28e30]. Therefore, the traffic across the secretory pathway was shown from GFP expression according Lippincott-Schwartz and Smith [31]. In order to evaluate the functionality of selected SPs, the presence and location of SP cleavage sites within the amino acid sequence of each propeptide were analyzed using the SignalP 4.1 server (Table 1). The last four codons of three SPs were changed by the corresponding AZ SP 3’codons. Modified SPs showed the same or even higher scores than wild type sequences. After this analysis, four vectors containing each SP followed by the GFP coding sequence (Fig. 1A) were con- structed. Two independent transfection experiments using CHO- K1, HEK293 and NS0 cells lines were performed with these con- structs. Fluorescence levels of cells transfected with GFP-containing vectors were evaluated by fluorescence microscopy (Fig. 2A) and measured by flow cytometry (Fig. 2B) 48 h after transfection. A SP- less construct (without SP) was used as expression control in cytoplasm. In consequence, the protein translation in the cytoplasm was a measurement of the highest GFP translation without the occurrence of the co-translational translocation limiting step. Considering only those cell lines transfected with plamids con- taining SPs, the GFP expression was higher when mSA SP was used as a signal sequence. Considering CHO-K1 cells, mSA SP showed fluorescence signals 5 to 7 times higher than AZ, mIgk C and mIgk H SPs. Also considering HEK293 cells, mSA SP fluorescence signals were 2e4 times higher, and for NS0 cells, they were more than 17 times higher than the same SPs. It is important to note that when using SP-constructs, lesser fluorescence signal within the cell was observed than when using a SP-less construct and it allowed concluding about the limiting step of the SPs-mediated secretory pathway. The fluorescence differences between SPs indicated the different efficiency of each SP, being the GFP signal highly propor- tional to mRNA levels [27].
Olczak and Olczak [32] observed that among the five heterolo- gous SPs that they studied in directly transfected insect cells, AZ SP was the most potent one. Kober el al [24]. described the AZ SP also as a sequence to improve production rates. Our results, in the assay conditions herein described showed that other SPs are better than AZ. In this sense, it is important to consider that the C-ter region of SPs has been modified with the corresponding region of the AZ SP, which might modulate its activity.
Most cell culture medium contains fluorescent compounds such as phenol red that represents a false-positive reagent for the mentioned method. Therefore, as the final step of a protein secre- tion, the presence of another protein easier to be detected in the culture medium as the chimera scFv-Fc was evaluated. For this purpose, an antigen specific indirect ELISA for the scFv Fc con- structs containing the same SPs employed for GFP was used.
To read the full manuscript, click here.
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