UTRtech™: Exploiting mRNA Targeting To Increase Protein Secretion From Mammalian Cells

Stern, Beate; Gjerdrum, Christine; Knappskog, Stian; Minsaas, Laura; Nylund, Stian; Olsen, Lene Christin; Olsen, Litta; Oveland, Eystein; Ravneberg, Hanne; Tröße, Christiane; Tveit, Anja; Vollsund, Endre; Hesketh, John E.; Tauler, Albert; Pryme, Ian F.

doi:10.1007/978-1-4020-5476-1_41

Cited by 3 publications

(4 citation statements)

References 2 publications

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“…The findings reported in this paper support those in previous studies (Knappskog et al 2007; Stern et al 2007a, b), where the efficiency of individual signal peptides was found to vary greatly. It was quite unexpected, though, to find that six of the seven oikosin signal sequences resulted in production of such strikingly low levels of luciferase activity when compared to the Gaussia luciferase signal peptide.…”

Section: Discussionsupporting

confidence: 92%

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Vectors Encoding Seven Oikosin Signal Peptides Transfected into CHO Cells Differ Greatly in MediatingGaussialuciferase and Human Endostatin Production although mRNA Levels are Largely Unaffected

Tröße¹,

Ravneberg²,

Stern

et al. 2007

Gene�Regul Syst Bio

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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.

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Section: Discussionsupporting

confidence: 92%

“…In a series of studies we have tested vectors encoding a variety of signal peptides for their efficiency with respect to synthesis and secretion of reporter proteins from transfected CHO cells (Knappskog et al 2007; Stern et al 2007a,b). It was found that the levels varied significantly.…”

Section: Introductionmentioning

confidence: 99%

Vectors Encoding Seven Oikosin Signal Peptides Transfected into CHO Cells Differ Greatly in MediatingGaussialuciferase and Human Endostatin Production although mRNA Levels are Largely Unaffected

Tröße¹,

Ravneberg²,

Stern

et al. 2007

Gene�Regul Syst Bio

Self Cite

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“…The protein structure prediction of the α-factor in the pre-region (Fig. 9 B) confirmed its prominent role and suggested that it forms an alpha helix that binds to signal recognition particles needed to enter the ER, as previously studied by Stern et al [ 62 ]. Meanwhile, the pro-region plays a vital role in the membrane translocation of the recombinant protein.…”

Section: Discussionsupporting

confidence: 76%

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Utami¹,

Nurdiani²,

Hariyatun³

et al. 2023

Journal of Genetic Engineering and Biotechnology

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Background Human insulin was the first FDA-approved biopharmaceutical drug produced through recombinant DNA technology. The previous studies successfully expressed recombinant human insulin precursors (HIP) in Pichia pastoris truncated and full-length α-factor recombinant clones. The matting α-factor (Matα), a signal secretion, direct the HIP protein into the culture media. This study aimed to compare the HIP expression from full-length and truncated α-factor secretory signals clones that grown in two types of media, buffered methanol complex medium (BMMY) and methanol basal salt medium (BSMM). Results ImageJ analysis of the HIP’s SDS-PAGE shows that the average HIP expression level of the recombinant P. pastoris truncated α-factor clone (CL4) was significantly higher compared to the full-length (HF7) when expressed in both media. Western blot analysis showed that the expressed protein was the HIP. The α-factor protein structure was predicted using the AlphaFold and visualized using UCSF ChimeraX to confirm the secretion ability for both clones. Conclusions CL4 clone, which utilized a truncated α-factor in the P. pastoris HIP expression cassette, significantly expressed HIP 8.97 times (in BMMY) and 1.17 times (in BSMM) higher than HF7 clone, which used a full-length α-factor secretory signal. This research confirmed that deletion of some regions of the secretory signal sequence significantly improved the efficiency of HIP protein expression in P. pastoris.

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“…The reason for that is their capacity to properly fold and assemble complex proteins and perform protein post‐translational modifications which are similar to human ones and are necessary for the stability, half‐life, and functionality of biopharmaceuticals . Even if productivity and yield of mammalian cell lines has increased dramatically in the past 2 decades reaching grams of recombinant protein per liter, there is still place for improving mammalian cells factories with regard to host cell line engineering, culture medium composition, vector optimization, protein secretion, yield, production cost, and potential virus contamination . In addition, CHO cell production appears to be expensive (50–300$ g −1 of product) and sufficient to supply only current markets concentrated in North America and Western Europe with a 14 day production run…”

Section: Introductionmentioning

confidence: 99%

Alga‐Made Anti‐Hepatitis B Antibody Binds to Human Fcγ Receptors

Vanier

Stelter

Vanier

et al. 2017

Biotechnology Journal

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Microalgae are unicellular eukaryotic organisms which represent an emerging alternative to other cell biofactories commonly used to produce monoclonal antibodies. Microalgae display several biotechnological advantages such as their rapid growth rate and their phototrophic lifestyle allowing low production costs as protein expression is solar-fueled. Recently, a fully assembled recombinant IgG antibody directed against Hepatitis B surface antigen is produced and secreted in the culture medium of the diatom Phaeodactylum tricornutum. A biochemical characterization of this recombinant antibody demonstrated that the Asn-297 is N-glycosylated by oligomannosides. In the immune system, antibodies interact with effector molecules and cells through their Fc part and the recognition of Fcγ receptors (FcγR) which are important for inducing phagocytosis of opsonized microbes. Interactions between IgG and FcγR are influenced by the N-glycan structures present on the Asn-297. In this study, the authors characterized the binding capacity of the anti-hepatitis B recombinant IgG produced in P. tricornutum to two human Fcγ receptors (FcγRI and IIIa) using a cellular binding assay and surface plasmon resonance (SPR). This allowed us to demonstrate that the alga-made antibody is able to bind FcγRI with a reduced affinity and engages FcyRIIIa with 3-times higher affinity compared to a control human IgG1.

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UTRtech™: Exploiting mRNA Targeting To Increase Protein Secretion From Mammalian Cells

Cited by 3 publications

References 2 publications

Vectors Encoding Seven Oikosin Signal Peptides Transfected into CHO Cells Differ Greatly in MediatingGaussialuciferase and Human Endostatin Production although mRNA Levels are Largely Unaffected

Vectors Encoding Seven Oikosin Signal Peptides Transfected into CHO Cells Differ Greatly in MediatingGaussialuciferase and Human Endostatin Production although mRNA Levels are Largely Unaffected

Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison

Alga‐Made Anti‐Hepatitis B Antibody Binds to Human Fcγ Receptors

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