Exploiting the ribosomal RPL10 R98S mutation to enhance mammalian recombinant protein production

Genomic screening studies recently revealed that samples from multiple cancer types contain somatic mutations in the ribosome. Ribosomal protein L10 (RPL10) carries an intriguing mutational hotspot, with the exact same substitution of arginine residue 98 (R98) to serine (R98S mutation) being present in 8% of children with T-cell leukemia. Strikingly, this residue is located at a key position within the ribosome and contacts the peptidyltransferase center. The effects of the RPL10 R98S mutation on cellular protein production were explored in a mouse pro-B cell model (Ba/F3) and a human T-ALL cell line (Jurkat) which were genetically engineered to obtain isogenic lines with or without the R98S mutation. We consistently detected 60-75% higher fluorescent signal upon expression of a GFP vector in R98S mutant Ba/F3 and Jurkat cell lines as compared to wild type cells. Moreover, chymotrypsin and caspase proteasomal activity was decreased by 25% in R98S mutant cells. The effects of R98S on ribosomal protein production rates are currently being explored. Translational fidelity was analyzed using dual-luciferase reporter assays evaluating correct reading of the genetic code (missense and nonsense reading). These assays indicated a 3- to 4-fold higher fidelity in the R98S Ba/F3 cells as compared to wild type cells. R98S mutant cells also showed enhanced cell survival in nutrient poor conditions which was associated with upregulation of pro-survival factor BCL-2. Polysomal qRT-PCR analysis showed a shift in Bcl-2 mRNA distribution towards the heavy polysome fraction in RPL10 R98S cells, supporting enhanced Bcl-2 translation efficiency by the R98S mutant ribosome. BCL-2 translation can be upregulated in cells under stress due to the presence of an internal ribosome entry site (IRES) motif within the Bcl-2 mRNA. Dual-luciferase reporter assays to evaluate IRES-dependent BCL-2 translation showed a 9-fold increase in BCL-2 IRES translation in RPL10 R98S Jurkat cells as compared to RPL10 wild type cells. The above described phenotypes of the R98S mutation may render this mutation of interest to enhance fidelity and efficiency of recombinant protein production in lymphoid cell lines, such as hybridomas, and in well-established industrial cell lines (Chinese Hamster Ovary (CHO) and Human Embryonic Kidney 293-T (HEK293-T)), especially when combined with a BCL-2 IRES motif in front of the recombinant protein coding sequence.

Authors

Benno Verbelen
Sergey O. Sulima
Tiziana Girardi
Sonia Cinque
Jonathan Royaert
Jelle Verbeeck
Stijn Vereecke
Kim R. Kampen
Kim De Keersmaecker

Organisations

KU Leuven Department of Oncology, Leuven, Belgium

Presenting author

Benno Verbelen, PhD student, KULeuven
benno.verbelen@kuleuven.be
Contact us now
REGISTER AS A DELEGATE NOW

Flanders.bio Strategic Partners

Flanders.bio Supporting Partners