The transfected cells were exposed to nnAA and the fluorescent proteins quantified by flow cytometry (Fig 3B)

The transfected cells were exposed to nnAA and the fluorescent proteins quantified by flow cytometry (Fig 3B). 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 VER 155008 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. Introduction Over the last ten years bioconjugates have emerged as a promising new class of medicines that offer improved performance of therapeutics. Bioconjugations have been used extensively to improve the half-life of proteins (e.g. PEGylation) and more recently for the construction of antibody drug conjugates (ADCs) [1,2]. ADCs are a promising new class of engineered biotherapeutic that combines the targeting specificity of antibodies with potent cytotoxins for the treatment of cancers. Early ADCs were generated by targeting cysteine thiols, or the primary amine of lysine for payload conjugation. However, this strategy generated heterogenous ADCs that have shown variable payload stability and reduced therapeutic effect [3,4]. To address these limitations technologies have emerged that enable site-specific modifications of target proteins to better control VER 155008 the homogeneity and stability of the products. One of the most attractive methods involves the site-specific incorporation of non-natural amino acids (nnAAs) containing moieties that enable biorthogonal conjugation chemistries [2,5C11]. Biorthogonal conjugation methods including oxime, Diels-Alder and click cycloaddition have all shown efficient conjugate formation and improved stability of the conjugates over the commonly used thiol-maleimide conjugations. The drawback to using nnAAs lies in the production of proteins containing these nnAAs. Expression systems in em E /em . em coli /em , yeast, mammalian cells (CHO and HEK293), and cell free expression systems have been developed and shown efficiency in nnAA incorporation [6, 8, 12C16]. Of these, mammalian cell expressions have the distinct advantage that they conform with conventional fermentation processes, contain a reliable glycosylation pattern, and are generally Mouse monoclonal to PPP1A devoid of endotoxin. Thus, great effort has been dedicated to developing cell lines that are capable of high titer expression of nnAA containing proteins. Stable cell lines expressing orthogonal aminoacyl tRNA synthetase/tRNA pairs have shown yields exceeding 1g/L [7, 17]. However, a good understanding of the minimal expression requirements of aaRS and its tRNA to enable efficient amber suppression is lacking. While the detection and quantification of aaRS expression is straightforward, the same cannot be said of tRNA. The measurement of tRNA is challenging due to its short size, and extensive secondary structure which restricts available qPCR probe sites. In addition, mature cytosolic tRNA, which averages 72 nucleotides is derived from a larger precursor molecule that undergoes 3 and 5 processing, and in eukaryotic cells is further modified with a 3 trinucleotide (CAA) to generate a functional tRNA [18]. Gel based methods requiring hybridization have been used to quantify the expression and assess the level of maturation of tRNAs [19C21]. While this method allows for the quantitation of aminoacylated, free, unprocessed and mature tRNA, it requires radioactive probes and is VER 155008 of low throughput. Molecular approaches to quantify specific tRNAs like four-leaf clover qPCR have been developed, but require large amounts of RNA, has a limited dynamic range, and requires multiple enzymatic steps [22]. We have developed a CHO-based expression system for the expression of biotherapeutics containing nnAAs [7]. The cells utilize the pyrrolysine tRNA synthetase (pylRS), derived from em Methanosarcina mazei /em , with specificity for a nnAA.