At present, the incorporation of non-natural amino acids has become an important problem that restricts heterologous protein expression. MAP (mussel adhesive protein), a medical adhesive, cannot be mass produced because
of the defect in the ability of aaRS to incorporate L-DOPA into protein. This causes the patient to choose sutures that cause more pain.
Why do we choose this project?
Background
MAP, a kind of protein complex secreted by mussel foot glands, is often used as biomedical adhesives because of its strong adhesion, good biodegradability and excellent biocompatibility. Therefore, they have a wide range
of prospects in medical and other industries1,2. L-3,4-Dihydroxyphenylalanine (L-DOPA), a noncanonical amino acid formed by hydroxylation of tyrosine, is widely distributed in natural MAPs, acting as a key
factor for adhesion
3,4. With the development of the medical industry, the demand for MAPs is rising. However, the extraction of natural MAPs is costly and inefficient. And thus microbial heterologous expression has become one
of the most important ways to produce this protein on a large scale5.
However, the natural aaRS cannot correctly increase L-DOPA into the protein, resulting in the loss of adhesion of MAPs producted by E. coli. Therefore, and we try to make aaRS more efficient and specific to recognize
L-DOPA using directed evolution strategies.
Previous treatments
Researchers have used many strategies to incorporate L-DOPA into MAPs. For example, Yang and co-workers used a tyrosine-auxotrophy strain to make endogenous TyrRS recognize the L-DOPA as much as possible by reducing one of the substrates of the TyrRS---tyrosine6.
The orthogonal translation system is appropriate for directly incorporating noncanonical amino acids such as L-DOPA7. Jeong and co-workers used Methanococcus jannaschii TyrRS (MjTyrRS) to incorporate L-DOPA
at a given stop codon to increase L-DOPA in MAPs8. However, due to the similarity between tyrosine and L-DOPA in structure, MjTyrRS cannot distinguish them well, which limits the efficiency of L-DOPA incorporation.
Our goal
Therefore, we start with MjTyrRS-Gen 29 (hereinafter referred to as "Gen 2"), an engineered TyrRS with improved L-DOPA-incorporating activity. And we try to make Gen 2 more efficient and specific to recognize
L-DOPA using directed evolution strategies.
Reference
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