Table 2 of
Gross, Mol Vis 2000;
6:40-50.Table 2 of
Gross, Mol Vis 2000;
6:40-50.
Table 2. Summary of Thirteen EcR1 Variants and Wild type EcR1
Codes used in the table: Y = Yes, N = No, blank = not tested, unknown. The number of pluses indicate the relative levels of expression, with 20+ indicating about 20 mg from 12 L of culture and one + indicating roughly 1 mg from 12 L of culture. For solubility 20+ indicates no visually observable turbidity at about 20 mM concentration, one + indicates no visually observed turbidity at about 1 mM protein concentration, intermediate numbers of pluses indicate no turbidity to visual inspection at 2 mM, the +/- indicates some evidence of turbidity at 1 mM. For fluorescence enhancement, 10+ indicates about a 10-fold enhancement of retinol fluorescence with 1 mM protein compared to the fluorescence of 1.5 to 3 mM free retinol, while "-" indicates no additional fluorescence over that of free retinol. Intermediate values of 0.5+ to 2+ indicate subjective relative fluorescence enhancement versus free retinol based on visual inspection of binding curves. Graphs for representative binding curves are presented in Figure 3. Fluorescence Enhancement means retinol fluorescence enhancement, WT CD indicates whether a protein was tested by CD scans and whether the conformation was grossly similar to EcR1. The antibody used (Ab) for detection on western blots was H3B5.
Expressed Soluble Relative Antibody WT Fluorescence
Mutant Substitution in E. coli in E. coli expression detection Solubility CD enhancement
------ ------------ ---------- ---------- ---------- --------- ---------- -- ------------
1 V116N N
2 L147A N
3 R148D N
4 G152A Y Y ++ Y ++ Y 2+
5 G153A N
6 L208A Y N + Y -
7 E218A Y N ++ Y/N ++ -
8 T237A Y N ++ Y + 0.5+
9 G239T Y N +++ Y ++ Y 2+
10 I249A Y N ++ Y + Y 1.5+
11 E251A Y N + Y ++ -
12 G278A Y N + Y ++ 1.5+
13 P281A Y N ++++ Y +/- 1+
EcR1 none Y Y 20+ Y 20+ Y 10+
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