Systematic use of synthetic 5'-UTR RNA structures to tune protein translation improves yield and quality of complex proteins in mammalian cell factories

• Predictably regulating protein expression levels to improve recombinant protein production has become an important tool, but is still rarely applied to engineer mammalian cells. We therefore sought to set-up an easy-to-implement toolbox to facilitate fast and reliable regulation of protein expression in mammalian cells by introducing defined RNA hairpins, termed 'regulation elements (RgE)', in the 5'-untranslated region (UTR) to impact translation efficiency. RgEs varying in thermodynamic stability, GC-content and position were added to the 5'-UTR of a fluorescent reporter gene. Predictable translation dosage over two orders of magnitude in mammalian cell lines of hamster and human origin was confirmed by flow cytometry. Tuning heavy chain expression of an IgG with the RgEs to various levels eventually resulted in up to 3.5-fold increased titers and fewer IgG aggregates and fragments in CHO cells. Co-expression of a therapeutic Arylsulfatase-A with RgE-tuned levels of the required helper factor SUMF1 demonstrated that the maximum specific sulfatase activity was already attained at lower SUMF1 expression levels, while specific production rates steadily decreased with increasing helper expression. In summary, we show that defined 5'-UTR RNA-structures represent a valid tool to systematically tune protein expression levels in mammalian cells and eventually help to optimize recombinant protein expression.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinsk bioteknologi (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Medical Biotechnology (hsv//eng)

Nyckelord

cerebroside sulfatase

immunoglobulin G

oxidoreductase

recombinant protein

SUMF1 protein

human

5' untranslated region

animal

biosynthesis

CHO cell line

conformation

Cricetulus

gene expression

gene expression regulation

gene vector

genetics

HEK293 cell line

human

inverted repeat

metabolism

procedures

protein engineering

protein synthesis

5' Untranslated Regions

Animals

Cerebroside-Sulfatase

CHO Cells

Genetic Vectors

HEK293 Cells

Humans

Inverted Repeat Sequences

Nucleic Acid Conformation

Oxidoreductases Acting on Sulfur Group Donors

Protein Biosynthesis

Recombinant Proteins

Publikations- och innehållstyp

ref (ämneskategori)

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