Co-translational Incorporation ofTrans-4-Hydroxyproline into Recombinant Proteins in Bacteria

Abstract

Trans-4-hydroxyproline (Hyp) in eukaryotic proteins arises from post-translational modification of proline residues. Because the modification enzyme is not present in prokaryotes, no natural means exists to incorporate Hyp into proteins synthesized in Escherichia coli. We show here that under appropriate culture conditions Hyp is incorporated co-translationally directly at proline codons in genes expressed in E. coli. Amino acids not specified by the genetic code are common in native proteins and can be essential to both structure and function. Proteins that contain novel non-natural amino acid analogues, furthermore, are of value in structure and function studies and may possess beneficial therapeutic properties. Noncoded amino acids in proteins arise either by enzymatic modification of a transfer RNA (tRNA) aminoacylated with one of the 20 coded amino acids (e.g. selenocysteine from serine) or by post-translational modifications. Reproduction of these reactions when recombinant proteins are being expressed in heterologous hosts is often either inefficient or not possible. Because of these difficulties, many proteins that contain noncoded amino acids are not available in quantities sufficient for detailed biophysical and biochemical studies.Current methodology to make proteins that contain noncoded amino acids in in vitro systems is limited to the production of relatively small amounts of protein. Use of in vitro acylated suppressor tRNAs to insert amino acid analogues at termination codons in genes translated in vitro results in sitespecific incorporation (1) but suffers from inefficiency and low yield. These problems occur in part because of limitations in producing acylated tRNA, constraints in achieving appropriate concentrations of other translational components, and variability in suppression efficiency. In vivo approaches that use an aminoacyl-tRNA synthetase with both altered amino acid and tRNA recognition and suppressor tRNAs are promising (2-4), but experimental hurdles inherent to this approach have not yet been fully overcome.In a general strategy to produce proteins containing novel amino acids, the use of DNA coding triplets to insert the amino acid analogue should be more efficient than use of termination codons. If an aminoacyl-tRNA synthetase is sufficiently promiscuous, it will aminoacylate cognate wild-type tRNA with an amino acid analogue, and the misacylated-tRNA can be used as usual by the translational machinery. This phenomenon has been exploited to produce proteins in prokaryotic systems that contain, for example, analogues of phenylalanine (5) and tryptophan (6). Two requirements must be met for success in these experiments: (1) the analogue must be acylated onto a tRNA at a demonstrable rate, and (2) the analogue must accumulate in the cell at concentrations high enough to give adequate acylation. The first requirement can, in theory, be met either by exploiting the promiscuity of a wild-type synthetase or through mutagenesis to alter the substrate specificity of a wild-type syntheta...

Hydroxylation-induced Stabilization of the Collagen Triple Helix

“…No collagen sequences with Gly-4(R)Hyp-4(R)Hyp tripeptide units have been identified. However, it was shown recently that 4(R)Hyp can be incorporated in both the Xaa and Yaa positions of a collagenlike molecule in Escherichia coli when the NaCl concentration is high (54). Our results show that comparable stabilities should be obtained for collagen-like peptides expressed in this system.…”

Organic fluorine as a polypeptide building element: in vivo expression of fluorinated peptides, proteins and proteomes

“…This is explained by former studies showing that the biosynthesis of endogenous low-affinity proline transporters can be up-regulated by hyperosmotic induction. 129,130 In their study, the authors also confirmed, that the yield of protein expression in the presence of NCAA depends on the host-cell physiology, model protein and experimental conditions by using a variety of analogs. Furthermore, Conticello and co-workers investigated the effects of the incorporated epimers on the self-assembly of the related polypeptides.…”

Recombinant expression of hydroxylated human collagen in Escherichia coli

“…The presence of Hyp at both positions x and y by contrast, further stabilized the triple helical conformation of the peptides (Berisio et al 2004). The detection of several Hyp residues at position x on various types of collagen makes it difficult to predict the positional impact of Hyp on the thermal stability of more complex polypeptides (Bann and Bachinger 2000;Buechter et al 2003;Song and Mechref 2013). Although early work demonstrated that…”