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Tyrosine sulfation is an important posttranslational modification that occurs in higher eukaryotes and is involved in cell-cell communication, viral entry, and adhesion. We describe a protocol for the heterologous expression of selectively tyrosine-sulfated proteins in E. coli through the use of an expanded genetic code that cotranslationally inserts sulfotyrosine in response to the amber nonsense codon, TAG. The components required for this process, an orthogonal aminoacyl-tRNA synthetase specific for sulfotyrosine and its cognate orthogonal tRNA that recognizes the amber codon, are encoded on the plasmid pSUPAR6-L3-3SY; and their use, along with a simple chemical synthesis of sulfotyrosine, is outlined in this protocol. Specifically, the gene for a protein of interest is mutated such that the codon corresponding to the desired location of tyrosine-sulfate is TAG. Cotransformation of an expression vector containing this gene and pSUPAR6-L3-3SY into an appropriate E. coli strain allows the overexpression of the site-specifically sulfated protein with high efficiency and fidelity. The resulting protein contains tyrosine-sulfate at any location specified by a TAG codon, making this method significantly simpler and more versatile than competing methods such as in vitro enzymatic sulfation, chemical sulfation, and peptide synthesis. Once the proper expression vectors are cloned, our protocol should allow the production of the desired sulfated proteins in less than one week.
Tyrosine sulfation is a key posttranslational modification (PTM) found in higher eukaryotes1, 2. Endogenous sulfation of tyrosine residues at protein interfaces generally enhances interaction strength and as a result, functions to modulate cell adhesion and ligand/receptor association3–5. Most notably, sulfation plays a central role in endogenous chemokine signaling6, 7 and consequently is involved in the entry of HIV through binding of gp120 to its sulfated coreceptor CCR5 or CXCR48–10, as well as the entry of malaria through binding the sulfated Duffy antigen receptor11, 12. It is likely that tyrosine sulfation has many additional biological functions that are as of yet uncharacterized, since sulfotyrosine is predicted to be present in over 2100 mammalian proteins1.
Despite their ubiquity, sulfated proteins are difficult to study due to the lack of general methods for their production2. For example, the solid phase synthesis of sulfated proteins is generally limited to smaller polypeptides and is complicated by the acid lability of sulfotyrosine's sulfate group. Other methods such as chemical sulfation and the in vitro enzymatic modification of proteins with sulfotransferases suffer from harsh reaction conditions, product heterogeneity, or the absence of site-selectivity beyond the limited patterns dictated by sulfation's PTM sequence determinants. Our recently reported method for the direct cotranslational incorporation of sulfotyrosine into proteins in E. coli, which allows for heterologous expression of sulfated proteins, circumvents these challenges13. This method makes use of an orthogonal tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair that selectively recognizes sulfotyrosine and inserts it site-specifically into proteins in response to the TAG (amber) codon. This system has been used to both structurally and biochemically analyze the role of tyrosine sulfation in the naturally sulfated protein hirudin13, 14, and to evolve “unnatural” selectively sulfated proteins with enhanced activities through directed evolution methods15. Here we report the construction of an optimized expression vector encoding the components necessary for expressing sulfated proteins and provide a detailed protocol for efficient expression of sulfated proteins.
The optimization of the sulfotyrosine incorporation expression vector followed a similar strategy to that recently reported for the expression of proteins containing unnatural amino acids that serve as NMR probes16. Specifically, we inserted an additional copy of the sulfotyrosine-specific synthetase (sTyrRS) gene into pSup-sTyrRS, which originally encoded a constitutively expressed sTyrRS, six copies of the orthogonal tRNA (two cassettes of three each), and a CmR marker13. The additional sTyrRS was placed under the independent control of the ara and T7 promoters to allow for inducible synthetase overexpression. This resulted in a final plasmid, pSUPAR6-L1-4SY, that contains six copies of the orthogonal tRNA and two copies of the corresponding orthogonal sTyrRS, one of which is inducible. An alternate version of this plasmid, which contains three instead of six copies of the orthogonal tRNA, was also created (pSUPAR3-L1-4SY) by deletion of one three-tRNA cassette. In addition to the reported sTyrRS synthetase, we also tested another sulfotyrosine-specific synthetase that was identified in the original selection13 (unpublished results). This synthetase differed from the reported one by a single conservative mutation, C159T. As with the sTyrRS synthetase, two plasmids were generated using this mutant in the same fashion. The final plasmids are termed pSUPAR3-L3-3SY and pSUPAR6-L3-3SY (Figure 1a).
Initial evaluation of these plasmids by shake flask overexpression of a FAS-TE (fatty acid synthase thioesterase domain) amber mutant (position Phe-2375-sTyr) in shake flasks with rich media gave high protein yields with all four plasmids, ranging from 93 mg/L/OD595 to 146 mg/L/OD595, where the highest purified yield was obtained from expression with the pSUPAR6-L3-3SY clone (Figure 2a and Anticipated Results). In addition to FAS-TE expression, we used pSUPAR6-L3-3SY for the expression of several other proteins including the doubly sulfated antibody Fab 412d (Figure 2b and Anticipated Results), which afforded purified yields of 0.20 mg/L (approximately 30% of the analogous wild-type yield), the two singly sulfated Fab 412d mutants, which afforded purified yields approaching 70% of wild-type yield depending on the site of sulfotyrosine incorporation, and over six other singly and doubly sulfated antibodies, where we achieved between 30–90% of wild-type yield. (Wild-type refers to expression of the corresponding protein where tyrosines are encoded in place of sulfotyrosines.) Additional proteins that have been successfully expressed by others and us using pSUPAR6-L3-3SY include m-TNF, sulfated peptides derived from anti-HIV antibodies (private correspondence w/ H. Choe and M. Farzan), and sulfated human trypsinogens (private correspondence w/ M. Sahin-Toth). Mass spectrometry of sulfated proteins often results in a subpopulation of the [M+H-80] peak, corresponding to cleavage of sulfate during ionization17. However, ionization conditions during ESI-LCMS analysis were mild enough to give the correct [M+H] mass for all sulfated proteins we expressed, with very little or no presence of a cleavage product [M+H-80] peak. The incorporation of sulfotyrosine with pSUPAR6-L3-3SY proceeds with good yield and the expected high fidelity.
We note that although our original pSup-sTyrRS gave good yields of sulfated proteins in glycerol minimal media (GMML), expression in 2YT rich media often resulted in prohibitively high amounts of truncated protein products (unpublished results). However, expression using pSUPAR6-L3-3SY – though difficult in GMML due its moderate toxicity – consistently gave high yields of sulfated proteins in rich media (2YT, TB, LB, or SOB). (This is presumably because the activity of the engineered sulfotyrosine-specific synthetase becomes limiting when cells achieve the higher growth and protein expression rates associated with rich media, requiring synthetase overexpression from pSUPAR6-L3-3SY to compensate.) Therefore, the primary advantage of pSUPAR6-L3-3SY over pSup-sTyrRS is the expression of sulfated proteins in rich media conditions, which are easy to use and, for certain applications, strictly necessary. The following protocol details the facile synthesis of sulfotyrosine18 (Figure 1b) and the simple and efficient recombinant expression of sulfated proteins.
Trifluoroacetic acid (Fisher); Caution: Corrosive.
Chlorosulfonic acid (Fluka); Caution: Corrosive.
Diethyl ether (Sigma-Aldrich); Caution: Flammable.
Standard LB agar (Fisher)
Standard 2YT, LB, SOB, or TB media (Fisher) or custom minimal media
DH10B (Invitrogen), Top10 (Invitrogen), Top10F' (Invitrogen), or BL21(DE3) (Novagen) cells
Qiagen Plasmid Mini Kit
Stratagene Quikchange II Mutagenesis Kit
pSUPAR6-L3-3SY or pSup-sTyrRS (both available from P.G.S.; ude.sppircs@ztluhcs)
Standard chemical synthesis supplies: round bottom flask, vacuum manifold setup with nitrogen line, stir bars, glass syringe, metal needles, Erlenmeyer flasks, filter funnels
Variable temperature shakers and incubators
NOTE: The amino acid sulfotyrosine is also commercially available from Bachem.
PAUSE POINT: The sulfotyrosine solution can be stored at 4 °C for at least 2 months.
PAUSE POINT: Plasmid mutants can be stored indefinitely at −20 °C.
Steps 1–6: 2 hours
Step 7: Overnight
Step 8–10: 3 days
Step 11–13: 3 days
Step 14–18: 2–3 days
Step 19–20: Can be done in parallel with steps 11–18
|6||Too much precipitant||Chlorosulfonic acid was old or too much moisture was introduced into the reaction, leading to hydrolysis of chlorosulfonic acid||Use fresh, dry reagents and ensure dry conditions for synthesis. Purity is not affected as the unreacted material is removed through filtration so this problem is tolerable.|
|18 and 19||Amber mutant yields protein in the absence of sulfotyrosine||Purification is problematic or there is a mutation in the expression vector||Background incorporation of natural amino acids in the absence of sulfotyrosine by our synthetases has never been detected in our experiments. Therefore, check that the expression vector contains the desired amber mutant, and that purification has removed all truncated protein that may complicate analysis.|
|18 and 20||Yield of sulfotyrosine-containing protein is undetectable, but yield of full-length protein with tyrosine encoded in the stead of sulfotyrosine is high||Mutation in a tRNA cassette promoter that disables expression of tRNA||Sequence the synthetase plasmid and if there is a mutation in the tRNA region, repeat expression from step 13, ensuring that the clone picked is one of the small majority colonies, not a rare outlier large colony.|
|18 and 20||Yield of sulfotyrosine-containing protein is consistently very low compared to yield of full-length protein with tyrosine encoded in the stead of sulfotyrosine||Suppression of amber codon is inefficient||Choose another site for sulfotyrosine incorporation if possible. If not, silently mutate the codons before and after the TAG codon as codon context of TAG has been shown to correlate to expression efficiency.|
|18 and 20||Yield of sulfotyrosine-containing protein and yield of full-length protein with tyrosine encoded instead of sulfotyrosine are both consistently low||Suboptimal expression conditions and temperature||Make sure saturation is achieved after 6–8 hours from induction. If it is not, the protein being expressed is likely toxic. In this case, it will be necessary to induce at a higher OD and express at lower temperatures. For optimal expression, saturation should occur at an OD595 greater than 2.0 in rich media. (In fact, we have observed, in some cases, a saturated OD595 of ~14.0 using TB growth media.)|
HK100 E. coli cells (a DH10B variant) containing pSUPAR6-L3-3SY and a FAS-TE expression vector16 were grown to OD595 = 0.8 at 37 °C. The cultures were then moved to 30 °C and sulfotyrosine was added. At OD595 = 1.34, the cells were induced with 0.2% L-arabinose and allowed to shake at 30 °C, 250 rpm, for an additional 19 hours to reach a final OD595 of 14.0. Cells were lysed and the sulfated FAS-TE, containing a C-terminal His-tag, was purified using a Ni-NTA column, yielding 2.237 g/L. PAGE gel analysis of the expressed protein is shown below (Figure 2a). ESI-LCMS analysis gave an [M+H] mass of 33238 Da ([M+H] calculated = 33237 Da).
Doubly sulfated antibody 412d and several sulfated mutant variants of 412d were expressed in Top10F' cells that contained an expression vector and pSUPAR6-L3-3SY. For protein production, cells were grown at 37 °C in TB media supplemented with sulfotyrosine and induced with 0.2% L-arabinose at OD595 = 1.0. At OD595 = 1.5, IPTG (1 mM) was added and the cells were shaken at 18 °C, 250 rpm, for an additional 36 hours to reach a final OD595 of 2.1–5.0. Periplasmic lysis and protein purification using a Protein G column gave pure doubly sulfated Fabs with yields around 0.2–0.25 mg/L. SDS-PAGE gel analysis of four variants expressed under these conditions are shown below (Figure 2b). ESI-LCMS analysis for all variants gave the expected masses; for example, the original 412d gave an [M+H] mass of 48823 Da ([M+H] calculated = 48822 Da). A control expression of 412d where tyrosines were encoded instead of sulfotyrosines gave an [M+H] mass of 48663 Da ([M+H] calculated = 48662 Da).
95% yield, white solid
λmax = 263 and ε = 224 M−1 cm−1 at pH 7.5, aqueous
1H-NMR (500 MHz, D2O) θ 3.13 (dd, J = 5.5 Hz, J = 14.5 Hz, 1H), 3.34 (dd, J = 9 Hz, J = 14.5 Hz, 1H), 4.01 (dd, J = 5.5 Hz, J = 9 Hz, 1H), 7.33 (m, 2H), 7.38 (m, 2H)
LCMS (ESI) for calculated C9H11NO6S (M + 1) 262.25, observed 262.2
C.C.L. thanks the Fannie and John Hertz Foundation and the National Science Foundation for predoctoral fellowships. This research was supported by the US National Institutes of Health (GM62159).