EGFP as a fusion partner for the expression and organic extraction of small polypeptides.
Green fluorescent protein (GFP) is widely used as an excellent reporter module of the fusion proteins. The unique structure of GFP allows isolation of the active fluorescent protein directly from the crude cellular sources by extraction with organic solvents. We demonstrated the stable expression of four short polypeptides fused to GFP in Escherichia coli cells, including antimicrobial cationic peptides, which normally kill bacteria. EGFP module protected fusion partners from the intracellular degradation and allowed the purification of the chimerical proteins by organic extraction. The nature of the polypeptide fused to GFP, as opposed to the order of GFP and the polypeptide modules in the fusion protein, influenced the efficiency of the described purification technique.
Skosyrev
Vitaly S
VS
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Pushchino Branch, Russian Academy of Sciences, Pushchino, Moscow 142290, Russia. vitaly@fibkh.serpukhov.su
Rudenko
Natalja V
NV
Yakhnin
Alexander V
AV
Zagranichny
Vasily E
VE
Popova
Lubov I
LI
Zakharov
Mikhail V
MV
Gorokhovatsky
Andrey Yu
AY
Vinokurov
Leonid M
LM
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D023181:Antimicrobial Cationic Peptides; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D023181:Antimicrobial Cationic Peptides; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D008164:Luminescent Proteins; D010455:Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; C057636:tachyplesin peptide, Tachypleus tridentatus; D049452:Green Fluorescent Proteins; C042572:sarcotoxin I protein, Sarcophaga peregrina; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein