Table 1.
Examples of NMR Studies Using the SET Approach
| Tag | Target Protein & Property | Reported Effect(s) | Notes and References |
|---|---|---|---|
| GB1 (6.2 kDa; pI=4.5) |
Mouse prion protein (mPrP) |
Increased the expression yield and solubility. |
GB1 was used to enhance the expression yield and the solubility of selected mPrP constructs. (Hornemann etal. 2009) |
| GB1basic (6.2 kDa; pI=8.0) |
HPV16 E6 constructs (1) E6 (17 kDa; pI=9.0) (2) E6N (8.9 kDa, pI=6.7) (3) E6C (7.5 kDa; pI=9.7) |
Improved solubility and sample stability of HPV-16 E6 protein. |
GB1basic is a GB1-mutant (D22N, D36R, and E42K) with a pI of 8.0. It was used to express basic target proteins to avoid aggregation. Use of the GB1basic tag allowed preparation of stable NMR samples of E6N and E6C at 2 mM, and E6 at 0.2 mM. The intrinsic solubility of E6N (after removing the GST-tag from GST-E6N) was in the range of hundreds of µM. (Liu etal. 2009) |
| GB1 (invisible C- terminal tag; 6.2 kDa; pI=4.5) |
Vav C-terminus SH3 (7.5 kDa; pI=6.4) |
Enhanced the solubility of VcSH3 by more than 10 fold. |
The target protein was initially expressed as an N- terminal GB1-fusion construct. A sortase-mediated protein ligation method was used to ligate a second, unlabeled GB1 to the C-terminus of the target protein. The N-terminal GB1 tag was subsequently removed by protease cleavage. The Vav C-terminus SH3 was almost insoluble at physiological pH. Using invisible C- terminal GB1 tag enabled preparation of stable NMR samples at 0.6 mM and subsequent structural determination (PDB: 2KBT). (Kobashigawa et al. 2009) |
| GB1 (6.2 kDa; pI=4.5) |
Borealin (1) Full length (31.3 kDa; pI=9.84) (2) residues13–92 (9.4 kDa; pI=5.48) |
Significantly improved the protein yield in the soluble fractions. |
(Zhou et al. 2009) |
| Calmodulin (CaM; invisible tag; 16.8 kDa, pI=4.1) |
Sterile alpha motif (SAM) from p63 (7.5 kDa; pI=5.8) |
Enhanced solubility by over 20-fold. |
The target protein was inserted between GST and the calmodulin binding peptide (CBP). The unlabeled calmodulin, which serves the role of a solubility- enhancement tag, was added to form a CBP-calmodulin complex. The N-terminal GST-tag was then removed by protease cleavage. (Durst et al. 2008) |
| GB1 (6.2 kDa; pI=4.5) |
17β-hydroxysteroid dehydrogenase type 1 (HSD17 β1). (homodimer with a molecular weight of 70 kDa) |
Increased sample stability. |
The fusion protein formed soluble aggregates at high concentrations, but maintained enzymatic activity to allow NMR-based inhibitor studies. (Ludwig et al.2008) |
| GB1 (6.2 kDa; pI=4.5) |
Potassium channel- interacting protein 4a (KChIP4a, residue 1–34; 3.7 kDa; pI=4.0) |
Enhanced solubility. | (Schwenk et al. 2008) |
| GB1 (c-terminal tag; 6.2 kDa; pI=4.5) |
CK2 substrate (XT111– 132; 2.4 kDa; pI=8.2) |
Enhanced solubility of fused peptide in live cells |
GB1 was used as a soluble carrier of a phosphorylation site and provided the solubility needed for recording spectra in live cells. (Selenko et al. 2008) |
| GB1 (6.2 kDa; pI=4.5) |
mRNA-decapping enzyme Dcp2 Nudix domain (17.3 kDa; pI=8.5) |
Enhanced solubility. | The untagged Nudix domain was only marginally soluble. The GB1-tagged protein (in the presence of Arg/Glu additives) was stable at 0.5 mM for several weeks. (PDB: 2JVB) (Deshmukh et al. 2008) |
| GB1 (6.2 kDa; pI=4.5) |
Eukaryotic translation initiation factor eIF5 (residues 241–405; 19.3 kDa; pI=5.2) |
Enhanced solubility. | (Reibarkh et al. 2008) |
| GB1 (6.2 kDa; pI=4.5) |
Parkin ubiquitin like domain mutant (UbldR42P) (8.8 kDa; pI=6.7) |
The GB1 tag was used to overcome the poor expression and degradation of the UbldR42P mutant; without the GB1 tag, the UbldR42P could not be isolated. (Safadi and Shaw 2007) |
|
| GB1 (6.2 kDa; pI=4.5) |
A ubiquitin variant found at the N-terminus of S27a in Giardia lamblia (GlUbS27A; 7.0 kDa; pI=4.7) |
Enhanced solubility/sample stability. |
No protein expression was observed with the His- or HA-tagged constructs. The GB1-tagged GlUbS27A was stable at 1 mM for about a week at 25 °C. (Catic et al. 2007) |
| GB1 (6.2 kDa; pI=4.5) |
Fas Death Domain (Fas-DD; 9.9 kDa; pI=8.7) |
Increased sample stability/solubility. |
The untagged Fas-DD had an intrinsic tendency to form soluble aggregates at physiological pH. (Ferguson et al. 2007) |
| GB1 (6.2 kDa; pI=4.5) |
Inositol 1,4,5- trisphosphate receptor (IP3R) intraluminal loop L3-2 (2.3 kDa; pI=6.3) |
Increased sample stability/solubility. |
No protein expression was observed with a His-tagged construct. (Kang et al. 2007) |
| GB1 (6.2 kDa; pI=4.5) |
Murine eIF4E (25 kDa; pI=5.8) |
Greatly enhanced solubility. |
(Untagged) mammalian eIF4E behaved poorly in solution. (Moerke et al. 2007) |
| Poly Arg or Lys peptide tags |
BPTI-22 (a BPTI variant containing 22 alanines) |
Enhanced solubility by 4–6 folds. |
(Kato et al. 2007) |
| GB1 (6.2 kDa; pI=4.5) |
SRp20 RNA recognition motif (RRM; 9.6 kDa; pI=6.6) |
Enhanced solubility. | Poor solubility of the untagged protein prevented NMR studies. The GB1-SRp20 RRM was stable at 1 mM, which enabled structural studies. (PDB: 2I38 & 2I2Y) (Hargous et al. 2006) |
| GB1 (6.2 kDa; pI=4.5) |
9G8 RNA recognition motif (9G8 RRM; 11.3 kDa; pI=9.6) |
Enhanced solubility. | Poor solubility of the untagged protein prevented NMR studies. The GB1–9G8 RRM (in the presence of Arg/Glu additives) was stable at 1 mM. Hargous et al. 2006) |
| GB1 (6.2 kDa; pI=4.5) |
UBA domain of human bone marrow stromal cells ubiquitin-like protein (BMSC-UbP; 4.8 kDa; pI=4.0) |
Dramatically enhanced the solubility. |
The untagged UBA domain readily precipitated in solution. The GB1-UBA was stable at 1 mM. (PDB: 2CWB) (Chang et al. 2006) |
| GB1 (6.2 kDa; pI=4.5) |
Rat ADAR2 double- stranded RNA binding domain (dsRBD; 24.3 kDa; pI=6.2) |
Improved protein expression and solubility. |
The untagged rat ADAR2 dsRBD12 (74–-301) had low solubility in common NMR buffers. The GB1-fusion protein was stable at 0.8 mM. (Stefl et al. 2005; Stefl et al. 2006) |
| GB1 (invisible tag) |
Chitin-binding domain | Not reported | Used an intein-based strategy to incorporate the unlabeled GB1 tag into isotopically labeled proteins. (Züger and Iwai 2005) |
| GB1 (6.2 kDa; pI=4.5) |
eukaryotic translation initiation factor 2 gamma (eIF2γ; 51 kDa; pI=8.7) |
Enhanced solubility | GB1 was used to enhance the solubility of eIF2γ to enable studies of its interaction with eIF2α (Ito et al. 2004) |
| GB1 (6.2 kDa; pI=4.5) |
Mutant myotoxin a (MyoP20G; 4.7 kDa; pI=9.5) |
Increased the expression yield and enhanced the refolding efficiency. |
Untagged protein refolded poorly. The GB1 tag was removed after refolding. (Cheng and Patel 2004) |
| GB1 (6.2 kDa; pI=4.5) |
Human Ki67 FHA domain (hNIFK; 5 kDa; pI=4.5) |
Increased the protein yield and sample stability. |
(Li et al. 2004) |
| GB1 (6.2 kDa; pI=4.5) |
NALP1 Pyrin domain (10 kDa; pI=5.9) |
Enhanced solubility by ∼100-fold. |
Untagged protein aggregated at concentrations above ∼10 µM. The GB1-tagged protein was stable at 1 mM (PDB: 1PN5). (Hiller et al. 2003) |
| GB1 (6.2 kDa; pI=4.5) |
Human T-cell leukemia virus 1 (HTLV-1) Tax40N (4.3 kDa; pI=6.0) |
Not reported | (Li et al. 2003) |
| GB1 (6.2 kDa; pI=4.5) |
eIF5B–CTD (16.7 kDa; pI=8.7) |
Enhanced solubility | (Marintchev et al. 2003) |
| MBP (40.7 kDa; pI=5.2) |
Integrin αIIbβ3 (MW of β3 is 5.5 kDa; pI=9.2) |
Enhanced solubility | (PDB: 1M8O). (Vinogradova et al. 2002) |