African Journal of Biotechnology

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Enhancing the productivity of soluble green fluorescent protein through methionine-residue specific consensus approach

N Soundrarajan, S Edwardraja, SG Lee, H Yun, N Ayyadurai


Protein sequences might have been evolved against different environmental pressures, which results in non-optimum properties in their stability, activity and folding efficiency. Directed evolution and consensus-based engineering of proteins are the protein engineering principles for the re-evolution of such natural proteins exhibiting non-optimal properties. Here, we propose an approach to improve the physical properties of target protein by engineering protein with new methionine residues. Our aim of this study was to investigate whether the physical property of protein can be improved by altering the negative effect caused by the introduction of additional methionine residue in the protein. First, we attempted to perform combinatorial mutagenesis of methionine residues of green fluorescent protein (GFP) using the consensus amino acids of conserved sequences. Each methionine residue in the internal sequence of GFP was combinatorially mutated by methionine or amino acid showing the highest frequencies in conserved sequences (I for M78, F for M88, Y for M153, V for M218, K for M233), and the mutants showing fluorescence were selected. Among the variants, the mutant of M218K showed an enhanced soluble expression in Escherichia coli. Our results indicate that it is possible to engineer protein by mutating methionine residues, specifically. We expect that the proposed approach can be exploited to enhance the expression of target protein in soluble form with avoiding the intensive labor of random mutagenesis and screening.

Key words: Protein sequence, GFP, directed evolution, consensus engineering, mutagenesis.
AJOL African Journals Online