Production and isotope labeling of antimicrobial peptides in Escherichia coli by means of a novel fusion partner that enables high-yield insoluble expression and fast purification.
A method is presented that allows efficient production of antimicrobial peptides in bacteria by means of fusion to the histone fold domain of the human transcription factor TAF12. This small fusion partner drives high-level expression of peptides and leads to their accumulation in an entirely insoluble form, thereby eliminating toxicity to the host. Using the antimicrobial peptide LAH4 as an example, we demonstrate that neither affinity purification of the TAF12 fusion protein nor initial solubilization of inclusion bodies in denaturing buffers is required. Instead, crude insoluble material from bacteria is directly dissolved in formic acid for immediate release of the peptide through chemical cleavage at a unique Asp-Pro site. This is followed by purification to homogeneity in a single chromatographic step. Because of the elevated expression levels of the histone fold domain and its small size (8 kDa), this straightforward purification scheme produces yields in excess of 10 mg active peptide per liter of culture. We demonstrate that TAF12 fusion allows expression of a wide range of antimicrobial peptides as well as efficient isotope labeling for NMR studies.
Vidovic
Verica
V
Institut de Chimie, Université Louis Pasteur, CNRS UMR 7177, 67070 Strasbourg, France.
Prongidi-Fix
Lydia
L
Bechinger
Burkhard
B
Werten
Sebastiaan
S
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007554:Isotopes; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D035381:TATA-Binding Protein Associated Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D023181:Antimicrobial Cationic Peptides; D004274:DNA, Recombinant; D007554:Isotopes; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; C470012:TAF12 protein, human; D035381:TATA-Binding Protein Associated Factors