Tetrazine Amino Acid Encoding for Rapid and Complete Protein Bioconjugation

Materials and reagents

Biological materials

Strains

1. BL21(DE3) (Thermo Fisher, catalog number: EC0114). This strain of E. coli is optimized for over-expression of target proteins under the T7 transcriptional promoter that is commonly found in standard pET vectors. This strain contains a genomic copy of the T7 RNA polymerase controlled by the lacUV5 promoter so that upon introduction with IPTG, the T7 polymerase is expressed and transcribes the target gene to produce high quantities of recombinant target protein. This strain contains Release Factor 1 (RF1), the protein responsible for terminating translation at TAG amber stop codons so that when encoding Tet2-Et at TAG codons of target proteins, truncated protein will be produced along with full-length Tet2-protein. To avoid co-purification of truncated protein with full-length protein, C-terminal purification tags are recommended. For proteins that self-assemble into homo-multimers (dimers, trimers, etc.), purification can be challenging due to the possible co-purification of truncated forms that are incorporated as subunits in the assembly. If using N-terminal purification/solubilization tags, additional purification steps may be needed to remove truncated protein species. See Troubleshooting #1 for more discussion.

2. B95(DE3) ΔAΔfabR (Addgene, catalog number: 197655). This strain is a robustly growing BL21(DE3) derivative that lacks release factor-1 (RF1), the protein responsible for translation termination at TAG codons, as well as a spontaneously mutated fabR gene [10]. Endogenous TAG stop codons in 95 genes were mutated to TAA or TGA to maintain cellular health and minimize unwanted readthrough due to RF1 knockout. This strain is preferred over BL21(DE3) for protein expression because by lacking RF1, TAG codon suppression by GCE machinery is more efficient, and the production of truncated protein (caused by early TAG site termination) is minimized. B95(DE3) ΔAΔfabR cells require lower concentrations of antibiotics to maintain normal growth rates.

3. DH10B (Thermo Fisher, catalog number: EC0113). This strain can be used for faithful propagation of plasmids and for cloning needs when users clone their genes of interest into their preferred plasmid backbone. Do not use this strain for protein expression. Though we have not explicitly tested all of them, other classical cloning strains of E. coli can be used in place of DH10b, including NEB 10-beta (New England BioLabs, catalog number: C3019H), DH5α (e.g., Thermo Fisher, catalog number: 18258012), NEB 5-alpha (New England BioLabs, catalog number: C2987H), or TOP10 (Thermo Fisher, catalog number: C404010).

Plasmids

1. pAJE3-E7 (Addgene, catalog number: 214359). Machinery plasmid for Tet2-Et incorporation that expresses a copy of the Methanocaldococcus jannaschii (Mj)-TyrRS-(E7)-Tet2-Et RS for faithful Tet2-Et incorporation as well as a copy of its cognate amber codon suppressing tRNA under constitutive lpp promoters. This plasmid confers spectinomycin resistance and harbors a recently developed high-copy synthetic origin of replication [1,11]. This synthetic origin of replication in pAJE plasmids can be stably propagated in cells that contain other plasmids having any of the standard or typical origins of replications, such as ColE1/pBR322/pMB1, p15A, and CDF origins, adding to the utility and versatility of this machinery plasmid.

2. pET28-sfGFP^WT (Addgene, catalog number: 85492). Expresses wild-type sfGFP control protein with C-terminal His6 tag, under a T7 transcriptional promoter, kanamycin resistance, and pBR322 origin of replication. sfGFP is expressed by the addition of IPTG or lactose.

3. pET28-sfGFP¹⁵⁰ (Addgene, catalog number: 85493). Same as above except the sfGFP gene contains a TAG amber stop codon at site N150. The TAG codon is used to direct the translational encoding of Tet2-Et.

4. pET28-[GOI]^WT (you must create). Expresses your wild-type protein of interest (POI). You can clone your POI into pET28 by removing the sfGFP gene via restriction digest with NcoI and XhoI enzymes and replacing it with your gene of interest (GOI) using standard cloning techniques (e.g., ligation, Gibson Assembly, or SLiCE [12]). For the reasons mentioned above, a C-terminal purification tag is preferred if using the BL21(DE3) strain for expression. For helpful tips on construct design, see Troubleshooting tip 1.

5. pET28-[GOI]^TAG (you must create). Expresses your POI with Tet2-Et encoded at a TAG codon. You can clone your POI into pET28 backbone by removing the sfGFP gene by restriction digest with NcoI and XhoI and replacing it with your gene of interest (GOI) using standard cloning techniques (e.g., ligation, Gibson Assembly, or SLiCE). Using site-directed mutagenesis, change the codon to TAG (the amber stop codon) where you intend to encode Tet2-Et into your POI. See section B for recommendations on TAG site selection.

Reagents

Essential reagents

1. Tryptone (e.g., VWR, catalog number: 97063-386)

2. Yeast extract (e.g., VWR, catalog number: 97064-368)

3. NaCl (e.g., VWR, catalog number: 97061-274)

4. α-D-glucose (e.g., VWR, catalog number: 97061-168)

5. α-lactose (e.g., VWR, catalog number: AAA11074-0B)

6. Glycerol (e.g., VWR, catalog number: BDH24388.320)

7. Na₂HPO₄ (sodium phosphate dibasic) (e.g., VWR, catalog number: 97061-586)

8. KH₂PO₄ (potassium monobasic) (e.g., VWR, catalog number: BDH9268)

9. K₂HPO₄ (potassium dibasic) (e.g., VWR, catalog number: 97062-234)

10. NH₄Cl (e.g., VWR, catalog number: 12125-02-9)

11. (NH₄)₂SO₄ (e.g., VWR, catalog number: 7783-20-2)

12. Na₂SO₄ (e.g., VWR, catalog number: 7757-82-6)

13. MgSO₄ (e.g., VWR, catalog number: 7487-88-9)

14. CaCl₂ᐧ2H₂O (e.g., VWR, catalog number: 10035-04-8)

15. MnCl₂ᐧ4H₂O (e.g., VWR, catalog number: 13446-34-9)

16. ZnSO₄ᐧ7H₂O (e.g., VWR, catalog number: 7446-20-0)

17. CoCl₂ᐧ6H₂O (e.g., VWR, catalog number: 7791-13-1)

18. CuCl₂ (e.g., VWR, catalog number: 10125-13-0)

19. NiCl₂ (e.g., VWR, catalog number: 7791-20-0)

20. Na₂MoO₄ᐧ2H₂O (e.g., VWR, catalog number: 10102-40-6)

21. Na₂SeO₃ (e.g., VWR, catalog number: 10102-18-8)

22. H₃BO₃ (e.g., VWR, catalog number: 10043-35-3)

23. FeCl₃ (e.g., VWR, catalog number: 7705-08-0)

24. L-arabinose (e.g., VWR, catalog number: 5328-37-0)

25. Kanamycin (e.g., VWR, catalog number: 75856-684)

26. Spectinomycin (e.g., VWR, catalog number: 89156-368)

27. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (e.g., Anatrace, catalog number: I1003)

28. Agar (e.g., VWR, catalog number: 97064-336)

29. Tet2-Et amino acid (see General note 4 for reagent availability)

30. N,N-Dimethylformamide (DMF) (VWR, catalog number: BDH1117-4LG)

31. sTCO-PEG₅₀₀₀ (see General note 4 for reagent availability)

32. Sodium dodecyl sulfate (SDS) (VWR, catalog number: JT4095-4)

33. Silicone emulsion, Antifoam B^® (VRW, catalog number: JTB531-5)

Reagents necessary only for defined auto-induction media

1. Glutamic acid, Na salt (e.g., VWR, catalog number: 56-86-0)

2. Aspartic acid (e.g., VWR, catalog number: 56-84-8)

3. Lysine-HCl (e.g., VWR, catalog number: 657-27-2)

4. Arginine-HCl (e.g., VWR, catalog number: 1119-34-2)

5. Histidine-HCl-H₂O (e.g., VWR, catalog number: 5934-29-2)

6. Alanine (e.g., VWR, catalog number: 56-41-7)

7. Proline (e.g., VWR, catalog number: 147-85-3)

8. Glycine (e.g., VWR, catalog number: 56-40-6)

9. Threonine (e.g., VWR, catalog number: 72-19-5)

10. Serine (e.g., VWR, catalog number: 56-45-1)

11. Glutamine (e.g., VWR, catalog number: 56-85-9)

12. Asparagine-H₂O (e.g., VWR, catalog number: 5794-13-8)

13. Valine (e.g., VWR, catalog number: 72-18-4)

14. Leucine (e.g., VWR, catalog number: 61-90-5)

15. Isoleucine (e.g., VWR, catalog number: 73-32-5)

16. Phenylalanine (e.g., VWR, catalog number: 63-91-2)

17. Tryptophan (e.g., VWR, catalog number: 73-22-3)

18. Methionine (e.g., VWR, catalog number: 63-68-3)

Solutions

Essential solutions

1. LB/agar media (see Recipes)

2. 2× YT media (see Recipes)

3. SOC media (see Recipes)

4. Kanamycin stock (see Recipes)

5. Spectinomycin stock (see Recipes)

6. 1 M IPTG (see Recipes)

7. 25× M salts (see Recipes)

8. Trace metal stock solution (5,000×) (see Recipes)

9. 50× 5052 solution (see Recipes)

10. Tet2-Et solution (see Recipes)

11. ZY media (see Recipes)

12. ZY non-induction (ZY-NIM) and auto-induction media (ZY-AIM) (see Recipes)

Solutions necessary only for defined auto-induction media

1. Aspartate [5% (w/v), pH 7.5] (see Recipes)

2. 18 amino acid mix (25×) (see Recipes)

3. Defined-NIM and AIM media (see Recipes)

1. LB/agar media (0.5 L)

   Reagent	Final concentration	Amount
   Tryptone	1 % (w/v)	5 g
   Yeast extract	0.5 % (w/v)	2.5 g
   NaCl	1.0 % (w/v)	5 g
   Agar	1.5 % (w/v)	7.5 g
   H2O	n/a	To 500 mL
   Total	n/a	500 mL

   1. After mixing reagents thoroughly, autoclave on standard liquid setting to sterilize. Note the agar will not go into solution until autoclaved.

   2. After autoclaving, gently swirl the bottle to ensure molten agar is evenly mixed.

   Notes:

   i. Store LB/agar bottle in a 55 °C oven and pour plates on an as-needed basis. LB/agar can be stored in molten form for ~2 weeks if sterility is maintained.

   ii. If an oven is not available, plates can be poured with antibiotics once LB/agar is sufficiently cooled to touch. Plates can be stored at 4 °C for up to a week.




2. 2× YT media (1 L)

   Reagent	Final concentration	Amount
   Tryptone	1.6 % (w/v)	16 g
   Yeast extract	1.0 % (w/v)	10 g
   NaCl	0.5 % (w/v)	5 g
   H2O	n/a	To 1000 mL
   Total	n/a	1 L

   1. After mixing reagents thoroughly, autoclave on the standard liquid setting to sterilize.

   2. After autoclaving, allow it to cool to room temperature before use.




3. SOC media (50 mL)

   Reagent	Final concentration	Amount
   2× YT media	n/a	49 mL
   1 M MgSO4	10 mM	0.5 mL
   40% (w/v) α-D-glucose	0.4% (w/v) or ~20 mM	0.5 mL
   Total	n/a	50 mL

   1. 1 M MgSO₄ can be made by mixing 12.3 g of MgSO₄·7H₂O in water up to 50 mL total volume. Adjust mass of MgSO₄ accordingly if using a salt with a different hydration status.

   2. 40% (w/v) α-D-glucose can be made by mixing 20 g of α-D-glucose with water up to 50 mL total volume. Mix thoroughly until glucose is dissolved. Gentle heating in a microwave may facilitate the dissolution of glucose.

   3. Sterilize MgSO₄, glucose, and 2× YT solutions individually by autoclaving. Allow each component to cool to room temperature and mix as indicated above. Maintain sterility while adding components together.

   4. It is easy to contaminate SOC. We suggest breaking this into 5 × 10 mL aliquots before use or making smaller batches. If sterility is maintained, SOC can be stored at room temperature indefinitely. It can also be stored at -20 °C but avoid repeated freeze/thaws.




4. Kanamycin stock (10 mL)

   Reagent	Final concentration	Amount
   Kanamycin	50 mg/mL	0.5 g
   H2O	n/a	To 10 mL
   Total	n/a	10 mL

   1. Sterilize by filtering with a 0.2 μm syringe-end filter.

   2. Store in 1 mL aliquots at -20 °C.




5. Spectinomycin stock (10 mL)

   Reagent	Final concentration	Amount
   Spectinomycin	100 mg/mL	1 g
   H2O	n/a	To 10 mL
   Total	n/a	10 mL

   1. Sterilize by filtering with a 0.2 μm syringe-end filter.

   2. Store in 1 mL aliquots at -20 °C.

   Note: Do not confuse spectinomycin with streptomycin. These antibiotics are not interchangeable.




6. 1 M IPTG (10 mL)

   Reagent	Final concentration	Amount
   IPTG	1 M	2.3 g
   H2O	n/a	To 10 mL
   Total	n/a	10 mL

   1. Sterilize by filtering with a 0.2 μm syringe-end filter.

   2. Store in 1 mL aliquots at -20 °C.




7. 25× M salts

   Reagent	25× concentration	Amount for 25×
   Na2HPO4	0.625 M	88.7 g
   KH2PO4	0.625 M	85.1 g
   NH4Cl	1.25 M	66.9 g
   Na2SO4	0.125 M	17.8 g
   H2O	n/a	to 1 L
   Total		1 L

   1. Add the above components to a 2 L beaker containing a magnetic stir bar. Add water up to 900 mL and mix until all components have dissolved. Add the remaining volume of water to reach 1 L.

   2. Weights indicated are based on anhydrous salts. If using hydrated phosphate salts, adjust the weights accordingly to maintain indicated molarities.




8. Trace metal stock solution (5,000×)

   Reagent	Concentration	Amount for individual 30 mL stocks
   	5,000×	1×
   CaCl2·2H2O	20 mM	4 µM	8.82 g
   MnCl2·4H2O	10 mM	2 µM	5.93 g
   ZnSO4·7H2O	2 M	2 µM	8.62 g
   CoCl2·6H2O	2 mM	0.4 µM	1.32 g
   CuCl2	2 mM	0.4 µM	807 mg
   NiCl2	2 mM	0.4 µM	777 mg
   Na2SeO3	2 mM	0.4 µM	1.03 g
   Na2MoO4·2H2O	2 mM	0.4 µM	1.45 g
   H3BO3	2 mM	0.4 µM	371 mg
   FeCl3	50 mM	10 µM	486 mg
   H2O	n/a		To 30 mL

   1. For each of the metals above (except FeCl₃), make individual stock solutions using the indicated masses and dissolve in Milli-Q water up to 30 mL of total volume. Autoclave each metal solution separately to sterilize. The FeCl₃ must be dissolved in 0.1 M HCl up to 30 mL of total volume and then filtered (through a 0.2 µm filter) to remove insoluble material and sterilize (do not autoclave).

   2. Once all individual stock solutions are prepared, add 500 µL of each stock solution (except FeCl₃) to 20.5 mL of sterile Milli-Q water. Then, add 25 mL of the FeCl₃ solution. The total volume should be exactly 50 mL.

   3. This stock solution might show minor precipitation over time but is stable at 15–25 °C for years.




9. 50× 5052 solution (500 mL)

   Reagent	Final concentration	Amount
   α-D-glucose	25 mg/mL	12.5 g
   α-lactose	100 mg/mL	50 g
   Glycerol	25% (v/v)	125 mL
   H2O	n/a	to 500 mL
   Total	n/a	500 mL

   1. Add the glucose, lactose, and glycerol components to roughly 300 mL of warm water in a 0.5 L beaker containing a magnetic stir bar. Mix until all solutions have dissolved. Additional heating may be required via microwave to encourage lactose dissolution (CAUTION: Remove magnetic stir bar before microwaving). Once fully dissolved, add the remaining volume of water to reach 500 mL.

   2. Autoclave on liquid cycle to sterilize.




10. Tet2-Et solution

    Reagent	Final concentration	Amount
    Tet2-Et	100 mM	8.5 mg
    DMF	n/a	to 275 µL
    Total	n/a	275 µL

    1. Vortex solution after combining to ensure all Tet2-Et has dissolved.

    2. Solution can be stored at -20 °C for months but may come out of solution and require additional vortexing upon freeze/thaw cycles. For optimal expressions, prepare the solution directly before use to avoid freeze/thaw cycles.




11. ZY media

    Reagent	Final concentration	Amount
    Tryptone	1% (w/v)	10 g
    Yeast extract	0.5% (w/v)	5 g
    H2O	n/a	to 1 L
    Total	n/a	1 L

    1. Add the above components to a 1 L beaker containing a magnetic stir bar. Add water up to 900 mL and mix until all solutions have dissolved. Add the remaining volume of water to reach 1 L.

    2. Autoclave for sterilization.




12. ZY non-induction (ZY-NIM) and auto-induction media (ZY-AIM)

    Reagent	ZY-NIM	ZY-AIM
    	Amount	Amount
    ZY media	47 mL	47 mL
    MgSO4	0.1 mL	0.1 mL
    25× M salts	2 mL	2 mL
    50× 5052	-	1 mL
    40% (w/v) α-D-glucose	0.625 mL	-
    Trace metal (5,000×)	10 µL	10 µL
    Total	50 mL	50 mL

    1. When preparing media, dilute the concentrated components into ZY media. Do not mix concentrated stocks and then dilute with ZY media.

    2. For BL21(DE3), the final concentration for spectinomycin and kanamycin should be 100 µg/mL and 50 µg/mL, respectively. For B95(DE3) expressions, the final concentration for spectinomycin and kanamycin should be 50 µg/mL and 25 µg/mL, respectively.

    3. Prepare immediately before use with a sterile technique.




13. Aspartate [5% (w/v), pH 7.5]

    Reagent	Final concentration	Amount
    Aspartate	5% (w/v)	50 g
    H2O	n/a	To 1 L
    Total	n/a	1 L

    1. Mix by placing a suitable magnetic stir bar in a 2 L beaker and add 900 mL of water to the graduated cylinder. While stirring, add the appropriate amount of L-aspartic acid and adjust pH to 7.5 with 8 M NaOH. Add the remaining volume of H₂O to bring the solution to the final volume of 1 L.

    2. Sterilize by autoclaving on liquid setting.




14. 18 amino acid mix (25×) (1 L)

    Reagent	Concentration	Amount for 25×
    	25×	1×
    Glutamic acid, Na salt	200 µg/mL	8 µg/mL	5 g
    Aspartic acid	200 µg/mL	8 µg/mL	5 g
    Lysine-HCl	200 µg/mL	8 µg/mL	5 g
    Arginine-HCl	200 µg/mL	8 µg/mL	5 g
    Histidine-HCl-H2O	200 µg/mL	8 µg/mL	5 g
    Alanine	200 µg/mL	8 µg/mL	5 g
    Proline	200 µg/mL	8 µg/mL	5 g
    Glycine	200 µg/mL	8 µg/mL	5 g
    Threonine	200 µg/mL	8 µg/mL	5 g
    Serine	200 µg/mL	8 µg/mL	5 g
    Glutamine	200 µg/mL	8 µg/mL	5 g
    Asparagine-H2O	200 µg/mL	8 µg/mL	5 g
    Valine	200 µg/mL	8 µg/mL	5 g
    Leucine	200 µg/mL	8 µg/mL	5 g
    Isoleucine	200 µg/mL	8 µg/mL	5 g
    Phenylalanine	200 µg/mL	8 µg/mL	5 g
    Tryptophan	200 µg/mL	8 µg/mL	5 g
    Methionine	200 µg/mL	8 µg/mL	5 g
    H2O	n/a	n/a	To 1 L
    Total	n/a	n/a	1 L

    1. Add 800 mL of water to a 1 L beaker, then add 5 g of each amino acid while stirring with a magnetic stir bar. Since some amino acids have trouble dissolving in solution, warming the water prior to adding the amino acids can aid in the dissolution process. It may take several hours for each component to fully dissolve. Finally, bring the volume to 1 L with water.

    2. Sterilize by filtration.

    3. Aliquot 45 mL of 25× 18-amino acid mix into sterile 50 mL conicals.

    4. Store aliquots at -20 °C. Thaw working aliquot as needed, which can be stored stably at 4 °C for several months provided sterility is maintained.




15. Defined-NIM and AIM media

    Reagent	NIM	AIM
    	Amount	Amount
    Aspartate [5% (w/v) pH 7.5]	2.5 mL	2.5 mL
    50× 5052	-	1 mL
    18 amino acid mix	2.0 mL	2.0 mL
    25× M salts	2.0 mL	2.0 mL
    MgSO4 (1 M)	100 µL	100 µL
    Glucose [40% (w/v)]	6.25 mL	-
    Trace metal solution (5,000×)	10 µL	10 µL
    Sterile H2O	36.64 mL	41.89 mL
    Total	50 mL	50 mL

    1. When preparing media, add the concentrated components to sterile H₂O, do not mix concentrated stocks, and then dilute with sterile H₂O.

    2. For BL21(DE3), the final concentration for spectinomycin and kanamycin should be 100 µg/mL and 50 µg/mL, respectively. For B95, the final concentration for spectinomycin and kanamycin should be 50 µg/mL and 25 µg/mL, respectively.

    3. Prepare immediately before use with a sterile technique.

1. 1.7 mL Eppendorf tubes (e.g., VWR, catalog number: 87003-294)

2. 100 mm plates (e.g., VWR, catalog number: 470210-568)

3. 500 mL graduated cylinder (e.g., VWR, catalog number: 470344-338)

4. 15 mL conical tubes (e.g., VWR, catalog number: 89126-798)

5. 50 mL conical tubes (e.g., VWR, catalog number: 89039-656)

6. 14 mL sterile culture tubes (e.g., VWR, catalog number: 60818-689)

7. 250 mL baffled flasks (e.g., VWR, catalog number: 89095-266)

8. Micro pipette tips 10 µL (e.g., VWR, catalog number: 76323-394)

9. Micro pipette tips 200 µL (e.g., VWR, catalog number: 76323-390)

10. Micro pipette tips 1,000 µL (e.g., VWR, catalog number: 76323-454)

11. 12% Mini-PROTEAN^® TGX^TM precast protein gels, 15 well, 15 µL (Bio-Rad, catalog number: 4561046)

12. Mini-PROTEAN^® Tetra companion running module (Bio-Rad, catalog number: 1658038)

13. Mini-PROTEAN^® Tetra vertical electrophoresis cell for mini precast gels, 4-gel (Bio-Rad, catalog number: 1658004)

14. Disposable PD-10 desalting column, with Sephadex G-25 resin, 1.0–2.5 mL samples (Cytiva, catalog number: 17085101)

15. TALON^® Superflow^TM (VWR, catalog number: CA71006-006)

16. Nalgene^® bottle-top sterile filter (Millipore Sigma, catalog number: Z358223-12EA)

Equipment

1. Autoclave capable of sterilizing liquid media and culturing materials at 121 °C and of a saturated steam pressure of 15 PSI

2. Expression equipment:

   1. Static incubator for growing LB/agar plates (set to 37 °C) (e.g., VWR, catalog number: 97025-630)

   2. Shaker incubator for growing liquid cultures (e.g., New Brunswick I26R, Eppendorf, catalog number: M1324-0004)

      i. Shaker should be able to rotate at 200–250 rpm.

      ii. Refrigeration is necessary for expressions below room temperature (< 25 °C).

      iii. Shaker deck should have clamps to hold 250 mL and 2.8 L Fernbach flasks.

   3. Optical density 600 nm spectrophotometer (e.g., Ultrospec 10, Biochrome, catalog number: 80-2116-30)

3. Fluorometer capable of reading sfGFP fluorescence (excitation 488 nm/emission 512 nm). Handheld fluorometers work well for routine fluorescence reads (e.g., PicoFluor from Turner Biosystems)

4. Freezer (-20 °C) for storing plasmids and antibiotics (e.g., Fisher Scientific, catalog number: 10-549-264)

5. Ice machine (e.g., Fisher Scientific, catalog number: 09-540-003)

Software and datasets

1. ImageJ

Procedure