Development of stable isotope and selenomethionine labeling methods for proteins expressed in Pseudomonas fluorescens.

Pseudomonas fluorescens is a robust protein expression system that is very well suited for high throughput protein expression for structural genomics studies. Since NMR spectroscopy and X-ray crystallography are both used by various investigators in structure elucidation studies, the availability of target proteins labeled with stable isotopes or selenomethionine is essential for the determination of protein structures. A completely defined medium for the expression and stable isotope labeling of proteins in P. fluorescens has been developed. The expression level of Bacillus thuringiensis Cry34 in the modified medium is comparable to that obtained in the original medium. In addition, more than 95% incorporation of 15N was obtained in Cry34 using 15N ammonium sulfate and the quality of the protein, as assessed by NMR analysis, is comparable to that made using commercial medium. High levels of selenomethionine (SeMet) incorporation in the Xenorhabdus nematophilus insecticidal protein XptA2 were also obtained in P. fluorescens using the defined medium, allowing development of a method for obtaining highly purified XptA2. The following observations were made when inhibitors of endogenous methionine biosynthesis were used in P. fluorescens culture when SeMet was substituted in XptA2: (I) there is little inhibition of cell growth or recombinant XptA2 expression in the presence of SeMet concentrations up to 300 mg/L in cell culture, (II) there was greater than 95% SeMet incorporation ratio in recombinant SeMet-labeled XptA2 (SeMet-XptA2) and the incorporation ratio is consistent and reproducible and (III) finally, purified SeMet-XptA2 possesses similar protein structure and insecticidal activity relative to the unlabeled counterpart XptA2 as shown by bioassay and differential scanning calorimetric analysis. The high SeMet incorporation should provide high accuracy and resolution in XptA2 phase determination by multiwavelength anomalous diffraction (MAD), indicating that P. fluorescens is an excellent expression host to produce SeMet-labeled proteins for structural study.

Large scale production of Bacillus thuringiensis PS149B1 insecticidal proteins Cry34Ab1 and Cry35Ab1 from Pseudomonas fluorescens.

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.