Part:BBa_K4968000
MSmtA4
MSmtA4, a mutant protein derived from four sites of SmtA mutation, has been codon-optimized. The mutation effectively enhanced the adsorption capacity of heavy metals.
Design
In order to enhance the surface affinity for heavy metal ions and improve the ability of the protein to chelate heavy metal ions near its surface, we designed a variant named MSmtA4 based on the bacterial metallothionein SmtA derived from Synechococcus PCC 7942. We placed particular emphasis on selecting mutation sites that would not affect the spatial structure and function of the protein. When choosing the amino acids for mutation, we prioritized those with a larger accessible surface area (ASA) and smaller root mean square fluctuation (RMSF) values (Saffar et al., 2015), along with a positive charge, to enhance electrostatic interactions. Therefore, we selected Arg 26, Lys 8, and Lys 22, and mutated them to Cys. This mutation effectively strengthened the protein's adsorption capacity for heavy metal ions, especially cadmium ions.
Mutation
In order to improve the expression efficiency of MSmtA4 in the target bacterial strain, we optimized its codons. Below are the DNA sequence changes in MSmtA4 before and after mutation:
MSmtA4 sequence before Mutation（Optimized SmtA-BBa_K4968001）:
1 ATGACTAGCA CGACCTTAGT TAAATGCGCT TGTGAACCGT GCTTATGCAA
51 TGTTGACCCC TCTAAGGCGA TCGATAGAAA CGGCCTTTAT TATTGCAGCG
101 AGGCCTGCGC TGACGGCCAC ACGGGTGGAT CTAAAGGGTG CGGGCACACT
151 GGTTGCAATT GTCACGGT
MSmtA4 sequence after Mutation:
1 ATGACATCCA CGACGTTGGT CTGTTGTGCT TGTGAACCAT GCCTTTGTAA
51 TGTGGACCCT AGCTGCGCGA TTGACTGTAA TGGACTGTAT TATTGCTCTG
101 AAGCGTGCGC AGACGGACAC ACCGGCGGAA GCTGCGGATG CGGCCATACA
151 GGTTGTAATT GTCACGGT
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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