Difference between revisions of "Part:BBa K4275010"

Revision as of 09:57, 10 October 2022

MHETase-t

MHETase-t is a dockerin-fused variant of free MHETase (BBa_K4275009). The enzyme shares all the common sequences in the CDS with the former, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of MHETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.

Usage and Biology

The artificially-designed MHETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E. coli's Cell-surface Nanobody3(Nb3). It is believed that the immobilization of the two enzymes (FAST-PETase-t and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.

Design consideration

1. The sequence of the first 565 amino acids is identical with the free MHETase (BBa_K4275009).

2. A 10 aa long GS linker (GGGGS)2 is appended following the Pro565 residue at the C' terminal of the original sequence.

3. The 79 amino acid long type-I dockerin domain is fused at the terminal of the GS linker, followed by a TEV site and a 8xhis affinity purification tag (HHHHHHHH).

4. DNA sequence is codon-optimized based on the codon-usage table of E.Coli Strain K12.MG1655.

Sequence and Features