<figcaptionstyle="width:70%;left:0;right:0;margin:auto;"><b>Figure 23.</b> The immobilization efficiency of different PETase constructs after 30mins incubation in 4°C. Immobilization efficiency= ([initial protein] - [protein in the washing buffer]) / [initial protein]. The protein concentration in the beginning solution and in the washing buffer was measured by Bradford assay. We load 500ug protein sample to each 20mg Celite545 silica beads for all constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch (Batch #2) for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch (Batch #3) for FAST-PETase.</figcaption>
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<p>As seen in Figure 23, the [FAST_PETase-linker-Car9] showed the highest immobilization efficiency over all the constructs (100%). And the [FAST_PETase-linker-L2NC] showed the lowest immobilization efficiency (11.93%). The Tri-PETase with silica tag shows low immobilization efficiency comparing to the FAST_PETase with silica tags after we corrected the data with empty control respectively.</p>
<p>The [C-terminal L2NC-tagged FAST-PETase] and [C-terminal L2NC-linker-tagged FAST-PETase] showed the immobilization efficiency (18.65% and 11.93%, respectively), lower than the empty control’s in the same batch. That may result from the lack of LacO sequence which caused low expression level of proteins comparing to inducible expression. Another possible reason might be the positively charged L2NC would interact with negatively charged PETase surface and inhibit the immobilization function.</p>
<p>The [C-terminal L2NC-tagged FAST-PETase] and [C-terminal L2NC-linker-tagged FAST-PETase] showed the immobilization efficiency (18.65% and 11.93%, respectively), lower than the empty control’s in the same batch. One possible reason might be the positively charged L2NC would interact with negatively charged PETase surface and inhibit the immobilization function.</p>
<p>We observed that the protein samples containing no silica-tag fusion proteins and untagged PETase both showed immobilization behaviour to the silica beads when they were not attached with any silica tags. Two main reasons may cause the results: </p>
<ol>
<li>The container we used to incubate protein with silica beads was 1.5ml Eppendorf tube, and when using it, the certain portion of silica beads always precipitated at the bottom during incubation. The proteins may be trapped in the silica beads precipitated at the bottom. And the unspecific interaction between immobilized PETase and the surrounding proteins may also contribute to the immobilization rate.</li>
