<div>The differential proteolytic susceptibilities of different types of
linkers joining the same two protein domains were investigated, to
identify linkers displaying the highest resistance to degradation by
proteases in the environment. Five linkers, namely <i>Rigid</i> [3
repeats of EAAAK], <i>Flexible</i> [two repeats of SGGGG],
<i>Nat-Full</i>, <i>Nat-Half</i> and <i>Nat-Quarter</i> [3 linkers of
42, 21, and 9 residues, respectively, derived from a naturally-occurring
Coh2-adjacent sequence] were used to fuse two thermostable, and
proteolytically-resistant, domains, namely Coh2 (from <i>C.
thermocellum</i> CipA), and BSX (from <i>Bacillus sp.</i> NG-27). The
produced fusions were examined for linker degradation, using
electrophoretic (SDS-PAGE) and chromatographic (size exclusion)
experiments, following different periods of storage at 4 ⁰C.
<i>Rigid, Nat-Full, Nat-Half</i> and <i>Nat-Quarter</i> were degraded,
and released free Coh2 and BSX. However, <i>Flexible</i> displayed a
remarkable resistance to both environmental proteases and Subtilisin A
(a non-specific protease). Our data suggests that <i>Flexible</i>
imparts freedom of movement to Coh2 and BSX, and then reciprocally
receives protection against proteolysis from Coh2 and BSX, through their
dynamic steric inhibition (or ‘batting away’) of protease approach.</div>