Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli

Materials and Reagents

Expression strain options

1. B95(DE3) ∆A ∆fabR ∆serB (Zhu et al., 2019). Available upon request.

2. BL21(DE3) ∆serB (Addgene # 34929) (Park et al., 2011; Rogerson et al., 2015)

   1. Neither strain has any plasmids in them, and they are sensitive to all antibiotics.

   2. The B95 ∆A ∆fabR ∆serB strain is RF1-deficient, and so little or no truncated protein is produced. Thus, N-terminal purification tags can be used with minimal concern of co-purifying truncated protein. This strain is highly preferred when (i) expressing homomultimeric proteins, (ii) C-terminal affinity tags hinder protein function, (iii) N-terminal solubility enhancing domains are needed (e.g., SUMO, GST, MBP, etc.), or (iv) seeking to achieve improved yields of multi-site pSer incorporation. These cells grow modestly, slower than the BL21 strain (described next), but yields from this strain are at least equivalent and in many cases higher than those of the BL21 strain (Zhu et al., 2019).

   3. The BL21 (DE3) ∆serB strain contains Release Factor 1 (RF1), so truncated protein is produced along with full-length phospho-protein. To avoid co-purification of truncated protein, C-terminal rather than N-terminal purification tags are strongly recommended (Rogerson et al., 2015). Furthermore, for proteins that self-assemble to homomultimers (dimers, trimers, etc.), purification can be problematic due to the possible co-purification of truncated forms that are incorporated as subunits in the assembly. This is especially a concern when the TAG codon is near the C-terminus.

   4. These strains can be made chemically competent using the Inoue method (Green and Sambrook, 2020). Aliquots of chemically competent cells can be stored at -80 °C for at least 3 years. Do not re-freeze aliquots.

   5. The C321 ∆A strain, a different RF1-deficient strain of E. coli that has been used for recombinant pSer protein expression (Pirman et al., 2015), grows more slowly, is not compatible with T7-based protein expression systems or auto-induction media, and therefore should not be used with this protocol.

      
      

Plasmids

1. pKW2-EFSep (Addgene #173897)

   Machinery plasmid for pSer incorporation, chloramphenicol resistance/pBR322 origin of replication (Rogerson et al., 2015). The pKW2-EFSep machinery plasmid has the same origin of replication as standard pET/pBad/pGEX vectors. Therefore, these vectors cannot be used to express target proteins with pSer, due to origin incompatibility with pKW2-EFSep. Target proteins must be expressed from vectors with a different origin of replication, such as p15a, CloDF, or RSF. The pRBC plasmid described below is recommended for target protein expression.

2. pRBC-sfGFP (Addgene #174075)

   Expresses sfGFP wild-type (wt) control protein with C-terminal His6 tag, under a T7 transcriptional promoter, and ampicillin resistance/p15a origin of replication (Zhu et al., 2019).

3. pRBC-sfGFP 150TAG (Addgene #174076)

   Same as above (including the C-terminal 6x-His tag), except for the sfGFP gene containing a TAG amber stop codon at site N150.

4. pRBC-[x] wild type (you must create)

   Expresses your protein of interest [x] or wild type. It is important, at least for initial tests, to clone your wild-type protein into the pRBC plasmid, to ensure it will express as expected.

5. pRBC-[x] TAG (you must create)

   Your protein of interest [x] with a TAG codon at the intended site (or sites) of pSer incorporation.

   
   

Media reagents and materials

1. LB/agar media (see Recipes)

2. ZY media (see Recipes)

3. Non-inducing ZY Media (see Recipes)

4. Auto-inducing ZY media (see Recipes)

5. SOC media (see Recipes)

6. 25× M-salts (see Recipes)

7. 1 M MgSO₄ (see Recipes)

8. 40% (w/v) glucose (see Recipes)

9. 50× 5052 solution (see Recipes)

10. 5,000× Trace metals solution (optional, see Recipes)

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

12. Yeast Extract (e.g., VWR, catalog number: 97064-368)

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

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

15. MgSO₄·7H₂O (e.g., VWR, catalog number: 97062-134)

16. Na₂HPO₄ (sodium dibasic) (e.g., VWR, catalog number: AA13437-30)

17. KH₂HPO₄ (potassium monobasic) (e.g., VWR, catalog number: BDH9268)

18. NH₄Cl (e.g., VWR, catalog number: 97062-050)

19. Na₂SO₄ or (NH₄)₂SO₄ (e.g., VWR, catalog number: BDH9302 or BDH9216)

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

21. α-D-lactose (e.g., VWR, catalog number: 36218.A3)

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

23. Ampicillin (e.g., VWR, catalog number: 97061-442)

24. Chloramphenicol (e.g., VWR, catalog number: 97061-244)

25. Ethanol (e.g., VWR, catalog number: 89125-188)

26. Antifoam B (e.g., J.T. Baker, catalog number: B531-05)

27. Sodium Fluoride (e.g., VWR, catalog number: 470302-540)

28. Sodium pyrophosphate (e.g., VWR, catalog number: JT3850-1)

29. Sodium orthovanadate (e.g., VWR, catalog number: BT219470-25G)

30. Phos-tag acrylamide (e.g., VWR, catalog number: 101974-086)

31. Phosphatase inhibitor cocktail mix (e.g., Sigma, catalog number: P2850)

32. Phos-tag agarose resin (e.g., Wako Chemicals, catalog number: AG-501)

33. 1.7 mL Eppendorf tubes (e.g., VWR, catalog number: 87003-294)

34. 100 mm plates (e.g., VWR, catalog number: 470210-568)

35. 50 mL conical tubes (e.g., VWR, catalog number: 89039-656)

36. 250 mL baffled flasks (e.g., VWR, catalog number: 89095-266)

37. 2.8 L baffled Fernbach flasks (e.g., Sigma, catalog number: CLS44232XL)

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

   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)

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

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

      3. 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. Cell Harvesting

   1. Centrifuge capable of speeds of at least 5,000 rcf and able to hold volumes commensurate with culture sizes (e.g., Beckman Coulter, model: Allegra 25R)

   2. Centrifuge bottles that can withstand centrifugal forces and hold the volume of cultures grown for harvesting cells. The type of bottles depends on the centrifuge and rotor used.

   3. Freezer (-80 °C), if cells are to be stored before protein is purified (e.g., VIP ECO ULT freezer, VWR, catalog number: 76305-596).

4. 42 °C water bath (e.g., VWR, catalog number: 76308-830)

5. 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).

Procedure