Incorporation of Norvaline at Leucine Positions in Recombinant Human Hemoglobin Expressed in Escherichia coli

Abstract

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the ␤-and di-␣-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNA Leu , similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

Discovery and Investigation of Misincorporation of Serine at Asparagine Positions in Recombinant Proteins Expressed in Chinese Hamster Ovary Cells

“…Second, Asn starvation studies have also shown (24,25) that the frequency of misreading of the Asn codon, AAU, is much higher (2-10-fold) than for the AAC codon, whereas we see no codon preference even though about two-thirds of the Asn codons in our proteins are AAU. Third, mischarging, as seen here, is not codon-related but results from the attachment of a different but structurally related amino acid to its cognate tRNA; misincorporation can be prevented by supplementing the culture medium with the cognate amino acid, Asn, in this case (11,26).…”

“…However, when Asn is not present in sufficient amounts, other amino acids, especially ones with the most similar structures and physical and chemical properties, will be misactivated so that biosynthesis can continue. The misincorporation of norvaline, a non-protein amino acid, at leucine positions in recombinant human hemoglobin when the ratio of norvaline to leucine in culture medium is high is a good example (11).…”

“…These yields are due mainly to improvements in host cell engineering, cell line selection, and culture medium optimization (4). However, it is well known that overexpressing recombinant proteins can lead to nutritional stresses in the host cells and that these stresses can markedly increase the frequency of random translational errors, resulting in a heterogeneous mixture of proteins (5)(6)(7)(8)(9)(10)(11). A variety of translational errors have been observed during overexpression of proteins in Escherichia coli, including frame shifts, premature truncation, read-through, leaky stop codons, and amino acid misincorporation (12)(13)(14)(15)(16).…”

Kinetic Partitioning between Synthetic and Editing Pathways in Class I Aminoacyl-tRNA Synthetases Occurs at Both Pre-transfer and Post-transfer Hydrolytic Steps

“…E. coli (Ec) LeuRS activates several noncognate amino acids in vitro, including isoleucine, valine, methionine, and norvaline (Nva) (21, 30 -33). Among these, norvaline is a nonproteinogenic amino acid that is of particular importance because it can be incorporated into recombinant proteins in place of leucine (34). It was recently demonstrated that the in vivo concentration of norvaline in the cell may rise to 1 mM under anaerobic growth conditions (35).…”

Mutational unmasking of a tRNA-dependent pathway for preventing genetic code ambiguity

“…Leucyl-tRNA synthetase (LeuRS) and two other closely related aaRSs [isoleucyl-(IleRS) and valyl-(ValRS) tRNA synthetases] that aminoacylate aliphatic amino acids have been well documented to misactivate and edit noncognate amino acids that structurally overlap with their respective substrates (10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The hydrolytic editing active site for each of these enzymes is located within homologous domains called the connective polypeptide 1 [CP1 (13,14,16,(19)(20)(21)(22)(23)(24)(25)(26)].…”