qPCR assays to quantitate tRNApyl and pylRS expression in engineered cell lines

• Published in: PloS one 2019-05, Vol.14 (5), p.e0216356-e0216356
• Main Authors: Garcia, Andrew, Roy, Gargi, Kiefer, Christine, Wilson, Susan, Marelli, Marcello
• Format: Article
• Language: English
• Subjects: Amino acids; Amino Acyl-tRNA Synthetases - biosynthesis; Amino Acyl-tRNA Synthetases - genetics; Animals; Antibodies; Assaying; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biology and life sciences; Biotechnology; Cell lines; CHO Cells; Cricetulus; Deoxyribonucleic acid; DNA; Dynamic range; Engineering; Engineering and Technology; Gene expression; Genes; Genetic Engineering; Lysine - analogs & derivatives; Lysine - metabolism; Methanosarcina - enzymology; Methanosarcina - genetics; Methods; Nucleotides; Polymerase chain reaction; Protein engineering; Protein structure; Proteins; Quantitation; Real-Time Polymerase Chain Reaction; Research and Analysis Methods; RNA; RNA, Transfer - biosynthesis; RNA, Transfer - genetics; Secondary structure; Transfer RNA; tRNA
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0216356

Non-natural amino acids (nnAA) contain unique functional moieties that greatly expand the available tool set for protein engineering. But incorporation of nnAAs requires the function of an orthogonal aminoacyl tRNA synthetase/tRNA pair. Stable cell lines expressing these components have been shown capable of producing gram per liter levels of antibodies with nnAAs. However, little has been reported on the genetic makeup of these cells. To gain a better understanding of the minimal requirements for efficient nnAA incorporation we developed qPCR methods for the quantitation of the key components. Here we describe the development of qPCR assays for the quantification of tRNApyl and pylRS. qPCR was chosen because it provides a large dynamic range, has high specificity for its target, and is a non-radioactive method used routinely for cell line characterization. Designing assays for tRNAs present challenges due to their short length (~72 nucleotides) and high secondary structure. These tRNA assays have a ≥ 5 log dynamic range with the tRNApyl assays being able to discern the mature and unprocessed forms of the tRNApyl. Cell line analysis showed tRNApyl was expressed at higher levels than the CHO-K1 endogenous Met and Phe tRNAs and that >88% of tRNApyl was the mature form.