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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Nat Protoc. Author manuscript; available in PMC 2010 August 25.
Published in final edited form as:
PMCID: PMC2928070
NIHMSID: NIHMS209179

Efficient expression of tyrosine-sulfated proteins in E. coli using an expanded genetic code

Abstract

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.

INTRODUCTION

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 association35. 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 CXCR4810, 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).

Figure 1
(Schultz). (a) Map of the optimized pSUPAR6-L3-3SY plasmid expressing the components necessary for translational incorporation of sulfotyrosine in response to the TAG codon. (b) Synthesis of sulfotyrosine.

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.

Figure 2
(Schultz) (a) FAS-TE protein (sTyr at position 2375) on a denaturing SDS-PAGE gel stained with Coomassie blue. (b) Sulfotyrosine-containing 412d and variants on a denaturing SDS-PAGE gel stained with Coomassie blue. Samples are: 412d with a modified sulfation ...

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.

MATERIALS

REAGENTS

L-tyrosine (Sigma-Aldrich)

Trifluoroacetic acid (Fisher); Caution: Corrosive.

Chlorosulfonic acid (Fluka); Caution: Corrosive.

Ethanol

Diethyl ether (Sigma-Aldrich); Caution: Flammable.

Chloramphenicol (Fisher)

NaOH (Fisher)

Standard LB agar (Fisher)

Standard 2YT, LB, SOB, or TB media (Fisher) or custom minimal media

Glycerol (Fisher)

Agarose (Invitrogen)

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)

EQUIPMENT

Standard chemical synthesis supplies: round bottom flask, vacuum manifold setup with nitrogen line, stir bars, glass syringe, metal needles, Erlenmeyer flasks, filter funnels

Electroporation cuvettes

Petri dishes

Shake flasks

Variable temperature shakers and incubators

Lyophilizer

PROCEDURE

Chemical synthesis of sulfotyrosine (1)

NOTE: The amino acid sulfotyrosine is also commercially available from Bachem.

  • 1
    In a dry round bottom flask containing a stir bar, dissolve 10 g (55 mmol) of L-tyrosine in 50 mL trifluoroacetic acid and cool to −10 °C.
  • 2
    While stirring under nitrogen gas, add 5 mL of chlorosulfonic acid over 2 minutes. CAUTION: Chlorosulfonic acid is extremely corrosive; use proper safety precautions, and use glass syringes with metal needles.
  • 3
    Allow reaction to stir at −10 °C for 5 minutes, and then quench the reaction by slowly adding 3 mL of ethanol and allow the reaction to stir at room temperature for 2 minutes, during which some precipitation may occur.
  • 4
    Add 175 mL diethylether to precipitate out sulfotyrosine and filter the precipitant.
  • 5
    Wash precipitant 3X with 75 mL diethylether and dry precipitant under vacuum.
  • 6
    Dissolve the precipitant in 0.5 M aqueous NaOH (~100 mL) and adjust the pH to 7.5 using additional NaOH. At this point, the dissolved material is sulfotyrosine and the small amount of precipitant is unreacted tyrosine and/or undesired side products. Remove the precipitant by filtration, collecting the dissolved sulfotyrosine.
  • 7
    Lyophilize the dissolved sulfotyrosine, redissolve the lyophilized product in 100 mL ddH2O, and filter off any additional precipitant that may appear. Take the UV absorbance to calculate the concentration of sulfotyrosine (λmax = 263, ε = 224 cm−1 M−1, pH 7.5; ε was determined by using a known concentration of sulfotyrosine purchased from Bachem). This solution of sulfotyrosine is ready to use without further purification. Any small amount of residual tyrosine, impurities, or epimerized amino acid will not affect expression since the sulfotyrosine-specific synthetases only recognize L-sulfotyrosine.

PAUSE POINT: The sulfotyrosine solution can be stored at 4 °C for at least 2 months.

Cloning and expression of sulfated proteins

  • 8
    Ensure that the expression vector containing the gene of interest is compatible with pSUPAR6-L3-3SY and pSup-sTyrRS, both of which encode chloramphenicol resistance and contain the P15A origin of replication.
  • 9
    Using site-directed mutagenesis, for example with the Quikchange kit (Stratagene) and associated protocols, replace the codon corresponding to the desired location of sulfotyrosine with TAG.
  • 10
    If sulfotyrosine incorporation at multiple sites is desired, repeat site-directed mutagenesis to add TAG codons at additional sites. Expression of multiply sulfated proteins will suffer from lower yields. We have efficiently expressed singly and doubly sulfated proteins (see Introduction), but have not yet tested expression with three or more sulfotyrosines.

PAUSE POINT: Plasmid mutants can be stored indefinitely at −20 °C.

  • 11
    Cotransform 5 ng of the expression vector along with 5 ng of plasmid pSUPAR6-L3-3SY (for expression in rich media) or pSup-sTyrRS (for expression in minimal defined media) into electrocompetent E. coli cells that do not have endogenous amber suppression activity. These include DH10B, Top10, Top10F', and BL21(DE3) strains, but do not include DH5α and XL-1Blue strains.
  • 12
    Recover the electroporated cells in 300 μL of 2YT media and incubate at 37 °C for 1 h. Plate dilutions of the recovery cultures on LB plates containing chloramphenicol (30 μg/mL) and the antibiotic corresponding to the resistance marker of the expression vector. Incubate the plates at 37 °C for 48 h if DH10B, Top10, or Top10F' cells are used and 30 h if BL21 cells are used.
  • 13
    Examine the plate corresponding to a dilution that gives many individual colonies (>100). There are sometimes a few colonies first visible between 12 and 24 hours that are much larger than the majority of remaining colonies. These few colonies correspond to mutants that have deleted or disabled some or all of the orthogonal tRNAs encoded by pSUPAR6-L3-3SY or pSup-sTyrRS as the uncharged orthogonal tRNAs are mildly toxic. However, the majority of colonies, which should be of uniform and small size, contain the correct pSUPAR6-L3-3SY or pSup-sTyrRS plasmid. Pick one of these colonies and expand it into 5 mL 2YT supplemented with chloramphenicol (30 μg/mL) and the antibiotic corresponding to the resistance marker of the expression vector. Let this culture grow for 24–36 hours at 37 °C and 250 rpm until saturation and create a glycerol stock of this saturated culture for future use.
  • 14
    From the saturated preculture, inoculate, at a ratio of 1:100, a shake flask of fresh rich media (2YT, LB, SOB, or TB) or minimal media containing selective antibiotics. Add sulfotyrosine to a final concentration of 10 mM and allow these cultures to grow at 37 °C and 250 rpm. If large expression volumes are desired, use the glycerol stock from step 13 to grow a sufficiently large overnight preculture from which the expression culture can be inoculated.
  • 15
    This step can be performed using option A, option B, or option C, depending on the E. coli strain and the plasmid, pSUPAR6-L3-3SY or pSup-sTyrRS, being used.
    1. For pSUPAR6-L3-3SY in strains DH10B, Top10, or Top10F': at OD595 = 1.0, induce the expression of the sulfotyrosine-specific aaRS by adding L-arabinose to a final concentration of 0.2 %.
    2. For pSUPAR6-L3-3SY in strain BL21(DE3): at OD595 = 1.0, induce the expression of the sulfotyrosine-specific aaRS by adding IPTG to a final concentration of 1 mM.
    3. For pSup-sTyrRS in any strain: no induction of the synthetase is necessary.
  • 16
    At OD595 =1.5 in the case of pSUPAR6-L3-3SY or at OD595 = 1.0 in the case of pSup-sTyrRS, induce the expression of the protein of interest from the expression vector. If expression of this protein is induced in the same manner as aaRS induction, then this additional induction step is not necessary.
  • 17
    Allow cells to grow for at least 20 h at the desired temperature, shaking at >250 rpm. Ideally, the temperature should be chosen such that the maximum saturation of the culture is reached between 6–8 hours. This will depend on the resource requirements and toxicity of the protein of interest being expressed. For example, in our expression of sulfated Fab antibodies whose production is relatively toxic (see Introduction), we conducted this step at 15–25 °C.
  • 18
    Harvest the cells by centrifugation and purify the full-length protein of interest using the desired methods. Characterize using ESI-LCMS and other desired methods. If the site of sulfotyrosine incorporation is near the C-terminus of the protein of interest, full-length sulfated protein and truncated protein, where termination at TAG occurs instead of sulfotyrosine incorporation, will be of similar size. In this case, purification using an anion or cation exchange column, depending on the protein expressed, may allow effective separation based on the charge of sulfotyrosine. Secondary purification may be unnecessary if a C-terminal purification tag is present on the protein of interest or if the site of sulfotyrosine incorporation is near the N-terminus.
  • 19
    If desired, repeat or conduct in parallel steps 14–18, but without the addition of sulfotyrosine to the media during expression. This control should result in a lack of any full-length protein expression, as the TAG codon(s) will not be suppressed.
  • 20
    If desired, repeat or conduct in parallel steps 11–18 with an expression plasmid that does not contain a TAG codon but instead encodes a tyrosine at the locations where sulfotyrosines are desired. This control should yield full-length protein without sulfotyrosine addition and can be used to optimize expression conditions or to compare yields. In this control, make sure to include pSUPAR6-L3-3SY or pSup-sTyrRS in the cells, even though these are not necessary for protein expression, because their presence will affect the determination of optimized conditions for expression.

TIMING

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

TROUBLESHOOTING

StepProblemPossible reasonSolution
6Too much precipitantChlorosulfonic acid was old or too much moisture was introduced into the reaction, leading to hydrolysis of chlorosulfonic acidUse 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 19Amber mutant yields protein in the absence of sulfotyrosinePurification is problematic or there is a mutation in the expression vectorBackground 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 20Yield of sulfotyrosine-containing protein is undetectable, but yield of full-length protein with tyrosine encoded in the stead of sulfotyrosine is highMutation in a tRNA cassette promoter that disables expression of tRNASequence 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 20Yield of sulfotyrosine-containing protein is consistently very low compared to yield of full-length protein with tyrosine encoded in the stead of sulfotyrosineSuppression of amber codon is inefficientChoose 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 20Yield of sulfotyrosine-containing protein and yield of full-length protein with tyrosine encoded instead of sulfotyrosine are both consistently lowSuboptimal expression conditions and temperatureMake 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.)

ANTICIPATED RESULTS

Sulfated FAS-TE

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 412d Fab and variants

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).

Sulfotyrosine (1)

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

ACKNOWLEDGEMENTS

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).

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