Part:BBa_K4719020
CBD-ProThr box-ArCE4A chitin deacetylase and cellulose binding domain fusion protein
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 432
Illegal NgoMIV site found at 961
Illegal AgeI site found at 417 - 1000COMPATIBLE WITH RFC[1000]
Introduction
Usage and Biology
We fused a cellulose binding domain connected to a linker to the N-terminus of deacetylase ArCE4A to ensure a higher degree of deacetylation. For protein purification 6x his-tag was added to the N-terminus of cellulose binding domain.Bacterial cellulose-chitosan copolymer has applications in the biomedicine field due to in vivo biodegradability by the lysosome. What is more, the bacterial cellulose-chitosan copolymer is a convenient platform for further modifications that would aid in solving the need for the promotion of tissue development. The uncovered amino groups are susceptible to enzymes catalyzing an addition of targeted organic chemistry groups. For instance, after modifying reaction conditions, deacetylase ClCDA BBa_K4719024 can propylate chitosan, which can later be used for click chemistry reactions [1]. In the future, specific targets like drugs or amino acids could be linked to the polymer, promoting the healing properties of the material [2].
Experimental characterization
Protein expression
Fusion with CBDcenA significantly decreased the solubility of deacetylases. To increase protein stability, we employed E. coli ArcticExpress (DE3) which is adapted for protein expression in lower temperatures. Investigating different biosynthesis conditions in ArcticExpress (DE3) revealed that induction at OD600 0.8 and growing the cells overnight at 16°C is optimal for fusion protein production.
Deacetylation enzymatic activity analysis with fluorescence microscopy
Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-ProThr box-ArCE4A 100 nM - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCL buffer. The samples were incubated for 14 h at 37° while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98°C.
For cellulose-chitosan copolymer generation from cellulose-chitin exopolymer we used chitin deacetylase CBD-ProThr box-ArCE4A. To determine if the deacetylation of our cellulose-chitin copolymer was successful, we used Alexa Fluor™ 405 NHS ester dye that specifically binds to free amino groups. On that account, only deacetylated copolymers should produce any fluorescent signal at this wavelength. To verify that our purified deacetylases are enzymatically active, at first we checked deacetylation activity on enzymes natural substrate - chitin.
Protein expression of recombinant deacetylase containing a new linker
A new linker BBa_K4719023 generated by our software was cloned into the pNIC-CH-6His-CBDCenA-ArCE4A backbone by Gibson assembly. Investigating different biosynthesis conditions in ArcticExpress (DE3) revealed that induction at OD600 0.8 and growing the cells overnight at 16°C is optimal for fusion protein production.
Deacetylation enzymatic activity analysis with fluorescence microscopy
Deacetylation was performed in a reaction with a final volume of 200 µL: 2 µL 1 mM CoCl2, deacetylase CBD-FRF-AnCDA 50 nM - 2µM and filling the remaining volume with 20mM HEPES-NaOH ph8, 150mM NaCL buffer. The samples were incubated for 14 h at 37° while shaking at 300 rpm, reaction was stopped by incubating for 3 min at 98°C. The fluorescence microscopy was performed under the same conditions as in the first iteration of this construct.
Growth burden
In order to work with E. coli for designing constructs and testing synthetic biology systems, the growth burden of said synthetic biology tools has to be measured. We performed growth burden evaluation by measuring OD600 for five hours of modified and unmodified E. coli DH5α. The composite of recombinant deacetylase CBD-ProThr box-ArCE4A did not inhibit the growth of E. coli as seen in Figure 1.
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