Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

Abstract

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.

Figure 1

The mammalian cell system for incorporating 3-iodo-l-tyrosine into proteins in response to amber codons. 3-Iodo-l-tyrosine (IY), present together with l-tyrosine (Y) in the growth medium, is taken up into the cell and is then attached, by its specific E.coli mutant TyrRS, to the B.stearothermophilus (B. s.) suppressor tRNA^Tyr. This tRNA carries this unnatural amino acid to the amber codon on the mRNA and incorporates it into a protein (alloprotein). On the other hand, the endogenous, mammalian tRNA^Tyr·TyrRS pair incorporates l-tyrosine into the proteins at the corresponding tyrosine codon.

Figure 2

The suppressor tRNA^Tyr derived from the E.coli tRNA₂^Tyr (left) and the B.stearothermophilus suppressor tRNA^Tyr (right). The nucleotides corresponding to the consensus internal promoter sequences (box A, TRGCNNAGY for positions 8–16 and G18G19; box B, GGTTCGANTCC for positions 52–62) (25) are circled. The base substitutions made in the E.coli tRNA to generate box A are shown by the arrows. Modified nucleotides found in these tRNAs, prepared from their native organisms, are 4-thiouridine (s⁴U), 2′-O-methylguanosine (Gm), 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A), 5-methyluridine (T), pseudouridine (ψ) and 1-methyladenosine (m¹A) (22).

Figure 3

Construction of a plasmid carrying nine tandem copies of the B.stearothermophilus suppressor tRNA^Tyr. Three copies of this suppressor tRNA^Tyr gene, represented by the arrow, were assembled, by ligation at the BstXI sites (BstXI-1 and BstXI-2), into a sub-cluster with the primer-binding sites (pbs1 and pbs2) at the ends. Three copies of this sub-cluster, each with the EcoRI (E), HindIII (H) or BamHI (B) sites, were then assembled and cloned within the EcoRI–BamHI sites of pBR322.

Figure 4

Western blots with the anti-FLAG antibody for the detection of amber suppression in CHO cells. (A) The ras gene (lane 1) or the ras(Am) gene (lanes 2–8), each with the FLAG tag, was introduced into the cells. The human suppressor tRNA^Tyr (lane 3) or the pair E.coli TyrRS and E.coli suppressor tRNA^Tyr (lane 4) was expressed in the cells. This enzyme also has the FLAG tag. The B.stearothermophilus suppressor tRNA^Tyr was expressed, together with the E.coli TyrRS (lanes 5 and 8) or without the enzyme (lane 7), or the E.coli TyrRS alone was expressed in the cells (lane 6). The B.stearothermophilus suppressor tRNA^Tyr was expressed from the plasmid carrying a single copy of its gene (lanes 5 and 7) and from that carrying nine copies (lane 8). A 2.5 µg aliquot of cell extract was subjected to SDS–PAGE in lane 1, while a 4-fold greater quantity of the cell extract was analyzed in lanes 2–8. The protein detected in all of the cell extracts (lanes 1–8) is an endogenous protein in CHO cells. (B) The epidermal growth factor receptor (EGFR) gene containing an amber codon in position 1068 (lanes 1 and 3) and the wild-type EGFR gene (lane 2), each with a FLAG tag, were introduced into CHO cells. The B.stearothermophilus suppressor tRNA^Tyr and the E.coli TyrRS pair were expressed in the cells (lane 3). The faint band migrating at the level of the E.coli TyrRS (lanes 1 and 2) was due to endogenous proteins that respond to the anti-FLAG antibody.

Figure 5

Western blots for the detection of amber suppression in the absence (A) and presence (B) of 3-iodo-l-tyrosine. The ras(Am) gene was introduced into CHO cells (all lanes). The wild-type E.coli TyrRS (lanes 1 in A and B) or TyrRS(V37C195) (lanes 2 in A and B) was expressed in the cells, along with the B.stearothermophilus suppressor tRNA^Tyr. The presence or absence of 3-iodo-l-tyrosine (IY) and the type of expressed TyrRS, wt for wild-type TyrRS and mut for TyrRS(V37C195), are indicated above the lanes.

Figure 6

LC-MS analyses of the ras and ras(Am) products. (A) The liquid chromatographic profiles, detected with a UV spectrometer, for the fragments from the ras(Am) products (chart a) and the ras products (chart b). (B) The mass chromatograms for the IY fragment (charts a and c) and the Y fragment (charts b and d) from the ras(Am) products (charts a and b) and the ras products (charts c and d). The Y fragment consists of residues 17–42, SALTIQLIQNHFVDEYDPTIEDSYRK, of the Ras protein. Tyr32, underlined in the sequence, was replaced by iodotyrosine in the IY fragment. (C) The tandem mass spectrum of the IY fragment. The partial sequence in the direction from the N-terminus to the C-terminus reads as Val, Asp, Glu, iodotyrosine and Asp in that order.

Figure 7

Western blots for detection of inducible amber suppression for 3-iodo-l-tyrosine incorporation into Ras protein. The ras(Am) gene was introduced into CHO-Y cells, together with the Bacillus suppressor tRNA^Tyr gene (lanes 1–3). Tetracycline was added to the growth medium, in the presence (lane 1) or the absence (lane 2) of 3-iodo-l-tyrosine, or neither was added to the medium (lane 3).