One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports.

Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.     

Purification, amino terminal analysis, and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling

Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.     

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

     

Analysis of seven genes from the eryAI –eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA, and lying between eryA and the gene eryK, which is known to encode the C-12 hydroxylase, has been sequenced and shown to contain seven additional open reading frames (ORFs 13–19). On the basis of sequence similarities, roles are proposed for several of these ORFs in the biosynthesis of the deoxysugar mycarose and the deoxyaminosugar desosamine. A chromosomal mutant carrying a deletion in ORF15 has been constructed and shown to accumulate 3-O-mycarosyl-erythronolide B, as expected for an eryC mutant. Similarly, a chromosomal mutant carrying a deletion in ORF16 has been constructed and shown to accumulate erythronolide B, as expected for an eryB mutant.     

Mechanical production of pellets for the application of entomopathogenic nematodes: factors that determine survival time of Steinernema glaseri

Applications of infective juveniles (IJ) of entomopathogenic nematodes (EPN) formulated in pellets are still limited. This is principally due to limited advances in the technology of formulation. We aimed to develop a new method of mechanical formulation through material flow and to analyse its effect on the survival time of encapsulated EPN by varying the granular materials, the components of the aqueous suspension, the age of the nematodes and by applying a surface coating (C) to the pellet. Three-day-old and two-month-old Steinernema glaseri IJ were encapsulated with different proportions of diatomaceous earth (DE) and attapulgite clay (AC). The aqueous suspension containing the nematodes was prepared with double distilled water (DDW), varying proportions of Opuntia ficus-indica mucilage (OM) or gelatin (GL), and a sunflower oil surface treatment. The pellets were stored at an average room temperature of 23 ± 6°C. The best results were obtained with the following proportions: 100DE:0AC and 50DE:50AC, using the OM suspension, three-day-old nematodes and a surface C, which resulted in an average of 14 days survival time. These results confirmed that the nematodes do not die during mechanical encapsulation and that the age of the IJ as well as the loss of moisture during storage at room temperature were the factors that decreased the survival of encapsulated EPN. It was concluded that it is necessary to use neonate IJ and to reduce the moisture transfer rate in the granular structure in order to delay the desiccation of the encapsulated nematodes.