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Expression, Purification and Analysis of Recombinant Proteins Responsible for the Functional Properties of a Tough Biological Glue
Author(s) -
Falconer Rebecca,
Smith Andrew
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.631.15
Subject(s) - recombinant dna , plasmid , protein purification , chemistry , complementary dna , microbiology and biotechnology , size exclusion chromatography , inclusion bodies , coding region , biology , biochemistry , dna , gene , enzyme
The slug Arion subfuscus secretes a defensive glue that is very strong despite being composed of 97% water. The overarching goal for analyzing this glue is to guide the development of a biomimetic hydrogel in hopes of replacing staples and stitches in surgical practice. Previous research has identified a group of 11 proteins (all around size 15kDa) that are unique to the glue and are integral for adhesion. The goal of this research was to express and purify recombinant versions of all of these proteins. We then tested whether the proteins could stiffen gels, and whether they formed oligomers. Ligation independent cloning was used to insert cDNA coding for each protein into a plasmid vector. The plasmid contained a 6His‐tag to facilitate purification, and the coding sequence for Protein G to decrease toxicity of the protein to the bacteria. After amplification and purification of the recombinant plasmids, the plasmids were used to transform an expression vector designed to facilitate disulfide bond formation (SHuffleT cells, NEB). After triggering overexpression, the proteins were isolated by binding to nickel resins. All eleven recombinant proteins were successfully purified in this way. Five of the proteins were analyzed. In preliminary results, they had the expected gel‐stiffening activity. Additionally, the recombinant proteins were analyzed by size‐exclusion chromatography to determine the proteins' tendency to oligomerize. The proteins primarily formed dimers at the pH of slug glue, with some trimer formation. Future work will test whether oligomerization is necessary for gel‐stiffening. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .