Cell-free production of biologically active polypeptides: application to the synthesis of antibacterial peptide cecropin.
An approach to preparative production of polypeptides, including uneasily testable, degradable, and toxic ones, is proposed on the basis of in vitro expression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The approach implies that a polypeptide of interest is synthesized as a fusion protein with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantitative monitoring of the polypeptide synthesis, as well as solubility and stability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated.
Martemyanov
K A
KA
Institute of Protein Research, 142292 Pushchino, Moscow Region, Russia.
Shirokov
V A
VA
Kurnasov
O V
OV
Gudkov
A T
AT
Spirin
A S
AS
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry; D000890:Anti-Infective Agents / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002474:Cell-Free System; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008969:Molecular Sequence Data; D010455:Peptides; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D020080:Tandem Repeat Sequences / Q000235:genetics; D014158:Transcription, Genetic
D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C070016:cecropin P1-LI