title: Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.
abstract: The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Nagasundarapandian
authors: N
authors: Soundrarajan
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Hye-Sun
authors: HS
authors: Cho
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Byeongyong
authors: B
authors: Ahn
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Minkyung
authors: M
authors: Choi
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Le Minh
authors: le M
authors: Thong
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Hojun
authors: H
authors: Choi
authors: Department of Infectious and Avian Diseases, Chonbuk National University, Iksan 570-752, Korea.
authors: Se-Yeoun
authors: SY
authors: Cha
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Jin-Hoi
authors: JH
authors: Kim
authors: College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Korea.
authors: Choi-Kyu
authors: CK
authors: Park
authors: College of Veterinary Medicine, Konkuk University, Seoul Seoul 143-701, Korea.
authors: Kunho
authors: K
authors: Seo
authors: Department of Animal Biotechnology, Konkuk University, Seoul 143-701, Korea.
authors: Chankyu
authors: C
authors: Park
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D003488:Cyanogen Bromide / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D018628:Gene Dosage; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics*; D002479:Inclusion Bodies / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008826:Microbial Sensitivity Tests; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D066329:Protein Aggregates; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development
chemical_list: D023181:Antimicrobial Cationic Peptides; C085474:PMAP-36; D066329:Protein Aggregates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C097849:buforin II; C094880:protegrin-1; D049452:Green Fluorescent Proteins; D003488:Cyanogen Bromide