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BACKGROUND The effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties(More)
Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a(More)
Twenty Fmoc-protected trinucleotide phosphoramidites representing a complete set of codons for the natural amino acids were chemically synthesized for the first time. A pool of these reagents was incorporated into oligonucleotides at substoichiometric levels to generate two libraries of variants that randomly carry either few or many codon replacements on a(More)
9-fluorenylmethoxycarbonyl (Fmoc) and 4,4'-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr(More)
A method to delete in-phase codons throughout a defined target region of a gene has been developed. This approach, named the codon-based random deletion (COBARDE) method, is able to delete complete codons in a random and combinatorial mode. Robustness, automation and fine-tuning of the mutagenesis rate are essential characteristics of the method, which is(More)
Current sequence-based approaches to identify enzyme functional shifts, such as enzyme promiscuity, have proven to be highly dependent on a priori functional knowledge, hampering our ability to reconstruct evolutionary history behind these mechanisms. Hidden Markov Model (HMM) profiles, broadly used to classify enzyme families, can be useful to distinguish(More)
Secretion of Escherichia coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with(More)
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