TABLE 2.
Modification of the C terminus of smRS-GFP influences whole-cell fluorescence and the level of fluorescent protein in recombinant H. volcanii
| Expression plasmid | smRS-GFP C terminusa | Amino acid tag (abbreviation)b | Relative protein level (%)c | Whole-cell fluorescenced |
|---|---|---|---|---|
| pJAM1023 | ...GITHGMDELYK | AANDENYALAA (ssrA) | UD | − |
| pJAM1030 | ...GITHGMDELYK | AANDENYALEE (ssrEE) | 6.6 ± 1.3 | + |
| pJAM1025 | ...GITHGMDELYK | AANDENYALDD (ssrDD) | 5.5 ± 0.6 | + |
| pJAM1031 | ...GITHGMDELYK | AANDENYALLL (ssrLL) | <0.5 | − |
| pJAM1032 | ...GITHGMDELYK | AANDENYALKK (ssrKK) | <0.5 | − |
| pJAM1048 | ...GITHGMDELYK | AANDDKDLSNN (ssr?) | 28.9 ± 5.5 | ++ |
| pJAM1033 | ...GITHGMDELYK | AANDENYAL (ssr9) | <0.5 | − |
| pJAM1051 | ...GITHGMDELYK | AANDENYA (ssr8) | <0.5 | − |
| pJAM1050 | ...GITHGMDELYK | AANDENY (ssr7) | <0.5 | − |
| pJAM1049 | ...GITHGMDELYK | AANDEN (ssr6) | <0.5 | − |
| pJAM1046 | ...GITHGMDELYK | AAN (ssr3) | <0.5 | − |
| pJAM1044 | ...GITHGMDELYK | A (ssr1) | 8.1 ± 1.9 | + |
| pJAM1020 | ...GITHGMDELYK | 100 ± 4.4 | ++++ |
a
C-terminal 11 residues of smRS-GFP.
b
Amino acid residues added to the C terminus of smRS-GFP. The abbreviations used to indicate the type of modification are in parentheses.
c
Protein level based on quantitative immunoblotting and expressed as a percentage relative to smRS-GFP. Compared to proteins that were present at levels that were less than 0.5% of the smRS-GFP level, the ssrA-tagged fusion protein was undetectable (UD).
d
Relative level of fluorescence as observed by fluorescence microscopy of colonies, where ++++ is the highest level and − is undetectable.