title: Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens.
abstract: The 14kDa (Cry34Ab1) and 44kDa (Cry35Ab1) binary insecticidal proteins are produced naturally by Bacillus thuringiensis PS149B1 as parasporal inclusion bodies. Here, we show production of these two insecticidal proteins in recombinant Pseudomonas fluorescens and their subsequent purification to near homogeneity to provide large quantities of protein for safety-assessment studies associated with the registration of transgenic corn plants. The gene sequence specific for each protein was expressed in P. fluorescens and fermented at the 75-L scale. For Cry34Ab1, the protein accumulated as insoluble inclusion bodies, and was purified by extraction directly from the cell pastes at pH 3.4 with a sodium acetate buffer, selective precipitation at pH 7.0, and differential centrifugation. For Cry35Ab1, the protein was extracted from the purified inclusion bodies with sodium acetate buffer (pH 3.5) containing 0.5M urea, followed by diafiltration. No chromatography steps were required to produce over 30g of lyophilized protein powder with purity greater than 98%, while retaining full insecticidal activity against Western corn rootworm larvae. The proteins were further characterized to assure identity and suitability for use in safety-assessment studies.
authors.affiliation: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA. khhuang@dow.com
authors.forename: Ke-Xue
authors.initials: KX
authors.lastname: Huang
authors.forename: Monty
authors.initials: M
authors.lastname: Badger
authors.forename: Keith
authors.initials: K
authors.lastname: Haney
authors.forename: Steve L
authors.initials: SL
authors.lastname: Evans
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002479:Inclusion Bodies / Q000737:chemistry; D007313:Insecta / Q000472:pathogenicity; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D003313:Zea mays / Q000235:genetics / Q000469:parasitology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis