Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli
大肠杆菌重组蛋白中磷酸丝氨酸的位点特异性插入
发布: 2022年11月05日第12卷第21期 DOI: 10.21769/BioProtoc.4541 浏览次数: 2537
评审: Petru-Iulian TrasneaSrujana Samhita YadavalliNoelia ForesiAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Jul 2019
Abstract
This protocol describes the recombinant expression of proteins in E. coli containing phosphoserine (pSer) installed at positions guided by TAG codons. The E. coli strains that can be used here are engineered with a ∆serB genomic knockout to produce pSer internally at high levels, so no exogenously added pSer is required, and the addition of pSer to the media will not affect expression yields. For “truncation-free” expression and improved yields with high flexibility of construct design, it is preferred to use the Release Factor-1 (RF1) deficient strain B95(DE3) ∆A ∆fabR ∆serB, though use of the standard RF1-containing BL21(DE3) ∆serB is also described. Both of these strains are serine auxotrophs and will not grow in standard minimal media. This protocol uses rich auto-induction media for streamlined and maximal production of homogeneously modified protein, yielding ~100–200 mg of single pSer-containing sfGFP per liter of culture. Using this genetic code expansion (GCE) approach, in which pSer is installed into proteins during translation, allows researchers to produce milligram quantities of specific phospho-proteins without requiring kinases, which can be purified for downstream in vitro studies relating to phosphorylation-dependent signaling systems, protein regulation by phosphorylation, and protein–protein interactions.
Graphical abstract:
Orthogonal translation system (正交翻译系统)
Phosphoserine (磷酸化)
Amber suppression (琥珀抑制)
Protein phosphorylation (蛋白磷酸化)
Recombinant protein (重组蛋白)
Background
Cellular signaling pathways are tightly regulated by serine phosphorylation, yet, historically, it has been very challenging to produce site-specifically and homogenously phosphorylated proteins in sufficient quantity for biochemical, biophysical, and structural characterization. Aspartate or glutamate have often been used to mimic the negative charge of phosphoserine (pSer) and phosphothreonine; however, due to the differences in charge and structure of Asp and Glu compared to pSer, both Asp and Glu commonly fail to recapitulate the effects of authentic phosphorylation. Kinases can be used to install phosphates on proteins, but the complexities of kinase specificity, as well as their need to be phosphorylated to be active, limit their utility to the few that can be recombinantly expressed in an active form and have well-defined substrates. Further discussion of these challenges is provided in our earlier publication, on which we base the protocol described here (Zhu et al., 2019).
Genetic code expansion (GCE) technologies have been developed for the translational installation of pSer in response to amber (TAG) stop codons. These technologies require a phosphoserine amino-acyl tRNA synthetase (SepRS), its cognate tRNA with an anti-codon that can suppress TAG codons (Sep-tRNACUA), and an EFTu variant (EFSep) engineered to deliver the pSer-amino-acylated tRNA to the ribosome (Park et al., 2011; Rogerson et al., 2015). Additionally, all pSer expression hosts have their serB gene knocked out, which causes free pSer amino acid to build up inside the cell to levels sufficient to feed the GCE machinery (Park et al., 2011). A few such ∆serB strains have been developed, and the choice of which to use is an important factor in the success of pSer protein expression. We recently created a healthy Release Factor-1 (RF1—the protein responsible for terminating translation at TAG codons) deficient strain of E. coli for pSer protein expression, called B95(DE3) ∆A ∆fabR ∆serB (Zhu et al., 2019). As reported by Zhu et al. (2019), by coupling this expression host with the efficient pSer GCE machinery created by Chin and colleagues (Rogerson et al., 2015), this expression system produces pSer proteins at higher yields than when using the parent RF1-containing BL21(DE3) ∆serB strain, does so without buildup of truncated protein, and has been shown to express proteins with up to five pSer residues incorporated. Because little or no truncation is observed, target proteins can be expressed with N-terminal affinity and solubility tags (e.g., SUMO, GST, MBP) without co-purification of truncated protein. This pSer protein expression chassis grows healthily (doubling time of ~40–45 min at 37 °C), and avoids misincorporation of natural amino acids caused by near-cognate suppression seen in other RF1 deficient pSer protein expression systems. Target proteins are expressed from the well-established T7 promoter expression system for high levels of protein production, and expression is compatible with auto-induction media. Hydrolysis of the pSer moiety by endogenous E. coli phosphatases during protein expression can be an issue, depending on target protein and site of incorporation, and so expression conditions and times may require additional optimization. Described below is the general workflow for expressing pSer proteins with B95(DE3) ∆A ∆fabR ∆serB, as well as with BL21(DE3) ∆serB, should users decide to use this strain.
Materials and Reagents
Expression strain options
B95(DE3) ∆A ∆fabR ∆serB (Zhu et al., 2019). Available upon request.
BL21(DE3) ∆serB (Addgene # 34929) (Park et al., 2011; Rogerson et al., 2015)
Neither strain has any plasmids in them, and they are sensitive to all antibiotics.
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).
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.
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.
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
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.
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).
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.
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.
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
LB/agar media (see Recipes)
ZY media (see Recipes)
Non-inducing ZY Media (see Recipes)
Auto-inducing ZY media (see Recipes)
SOC media (see Recipes)
25× M-salts (see Recipes)
1 M MgSO4 (see Recipes)
40% (w/v) glucose (see Recipes)
50× 5052 solution (see Recipes)
5,000× Trace metals solution (optional, see Recipes)
Tryptone (e.g., VWR, catalog number: 97063-386)
Yeast Extract (e.g., VWR, catalog number: 97064-368)
NaCl (e.g., VWR, catalog number: 97061-274)
Agar (e.g., VWR, catalog number: 97064-336)
MgSO4·7H2O (e.g., VWR, catalog number: 97062-134)
Na2HPO4 (sodium dibasic) (e.g., VWR, catalog number: AA13437-30)
KH2HPO4 (potassium monobasic) (e.g., VWR, catalog number: BDH9268)
NH4Cl (e.g., VWR, catalog number: 97062-050)
Na2SO4 or (NH4)2SO4 (e.g., VWR, catalog number: BDH9302 or BDH9216)
α-D-glucose (e.g., VWR, catalog number: 97061-168)
α-D-lactose (e.g., VWR, catalog number: 36218.A3)
Glycerol (e.g., VWR, catalog number: BDH24388.320)
Ampicillin (e.g., VWR, catalog number: 97061-442)
Chloramphenicol (e.g., VWR, catalog number: 97061-244)
Ethanol (e.g., VWR, catalog number: 89125-188)
Antifoam B (e.g., J.T. Baker, catalog number: B531-05)
Sodium Fluoride (e.g., VWR, catalog number: 470302-540)
Sodium pyrophosphate (e.g., VWR, catalog number: JT3850-1)
Sodium orthovanadate (e.g., VWR, catalog number: BT219470-25G)
Phos-tag acrylamide (e.g., VWR, catalog number: 101974-086)
Phosphatase inhibitor cocktail mix (e.g., Sigma, catalog number: P2850)
Phos-tag agarose resin (e.g., Wako Chemicals, catalog number: AG-501)
1.7 mL Eppendorf tubes (e.g., VWR, catalog number: 87003-294)
100 mm plates (e.g., VWR, catalog number: 470210-568)
50 mL conical tubes (e.g., VWR, catalog number: 89039-656)
250 mL baffled flasks (e.g., VWR, catalog number: 89095-266)
2.8 L baffled Fernbach flasks (e.g., Sigma, catalog number: CLS44232XL)
Equipment
Autoclave capable of sterilizing liquid media and culturing materials at 121 °C, and of a saturated steam pressure of 15 PSI.
Expression
Static incubator for growing LB/agar plates (set to 37 °C) (e.g., VWR, catalog number: 97025-630)
Shaker incubator for growing liquid cultures (e.g., New Brunswick I26R, Eppendorf, catalog number: M1324-0004)
Shaker should be able to rotate at 200–250 rpm
Refrigeration is necessary for expressions below room temperature (< 25 °C)
Shaker deck should have clamps to hold 250 mL and 2.8 L Fernbach flasks
Optical density 600 nm spectrophotometer (e.g., Ultrospec 10, Biochrome, catalog number: 80-2116-30)
Cell Harvesting
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)
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.
Freezer (-80 °C), if cells are to be stored before protein is purified (e.g., VIP ECO ULT freezer, VWR, catalog number: 76305-596).
42 °C water bath (e.g., VWR, catalog number: 76308-830)
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
文章信息
版权信息
© 2022 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhu, P., Mehl, R. A. and Cooley, R. B. (2022). Site-specific Incorporation of Phosphoserine into Recombinant Proteins in Escherichia coli. Bio-protocol 12(21): e4541. DOI: 10.21769/BioProtoc.4541.
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分类
生物工程 > 合成生物学
生物工程 > 生物医学工程
生物化学 > 蛋白质 > 翻译后修饰
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