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Optimization of Protein Fusion Partner Length for Maximizing in Vitro Translation of Peptides
Author(s) -
Loose Christopher R.,
Langer Robert S.,
Stephanopoulos Gregory N.
Publication year - 2007
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp060277o
Subject(s) - proteolysis , amino acid , peptide , fusion , yield (engineering) , translation (biology) , protease , fusion protein , biochemistry , proteases , chemistry , in vitro , molar concentration , recombinant dna , enzyme , biology , messenger rna , organic chemistry , materials science , philosophy , linguistics , metallurgy , gene
Using protein fusion partners for in vitro translation may increase solubility, assist in purification, or allow detection of small proteins and peptides. Here we show that the molar yield of peptide in a batch reaction may be maximized by optimizing the length of the translated product, which is composed of the fusion partner plus the peptide. Using truncated versions of GFP as a series of fusion partners, the molar yield increased approximately 3‐fold as the length of the translated product was reduced from 250 to 100 amino acids. When the translated product was shortened below roughly 100 amino acids, molar yield fell as a result of proteolysis. This trend was verified using two fusion partners with different amino acid sequences. Furthermore, protease inhibitors were used to confirm that proteases were responsible for limiting accumulation of peptides below the optimal length.