Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and β-mercaptoethanol

The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and β-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20mM β-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90mg/l of the culture or 11.8 and 9.37mg/g of wet weight of cells in pQE30UA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE30UA as compared with purification by conventional methods.     

A public genome-scale lentiviral expression library of human ORFs

Functional characterization of the human genome requires tools for systematically modulating gene expression in both loss-of-function and gain-of-function experiments. We describe the production of a sequence-confirmed, clonal collection of over 16,100 human open-reading frames (ORFs) encoded in a versatile Gateway vector system. Using this ORFeome resource, we created a genome-scale expression collection in a lentiviral vector, thereby enabling both targeted experiments and high-throughput screens in diverse cell types.     

Enhanced Polar Auxin Transport in Tomato Polycotyledon Mutant Seems to be Related to Glutathione Levels

Glutathione-dependent root growth in Arabidopsis is linked to polar auxin transport (PAT). Arabidopsis mutants with reduced glutathione (GSH) levels also show reduced PAT. To gain an insight into the relationship between PAT and GSH level, we analyzed tomato polycotyledon mutant, pct1-2, which has enhanced PAT. Microarray analysis of gene expression in pct1-2 mutant revealed underexpression of several genes related to glutamate and glutathione metabolism. In consonance with microarray analysis, enzymatic as well as in vivo assay revealed higher glutathione levels in the early phase of pct1-2 seedling growth than WT. The inhibition of auxin transport by 2,3,5-triiodobenzoic acid (TIBA) reduced both GSH level and PIN1 expression in pct1-2 root tips. The reduction of in vivo GSH accumulation in pct1-2 root tips by buthionine sulfoximine (BSO) stimulated elongation of the short root of pct1-2 mutant akin to TIBA. The rescue of the short root phenotype of the pct1-2 mutant was restricted to TIBA and BSO. The other auxin transport inhibitors 1-N-naphthylphthalamic acid (NPA), 2-[4-(diethylamino)-2-hydroxybenzoyl] benzoic acid (BUM), 3-chloro-4-hydroxyphenylacetic acid (CHPAA), brefeldin and gravacin inhibited root elongation in both WT and pct1-2 mutant. Our results indicate a relationship between PAT and GSH levels in tomato akin to Arabidopsis. Our work also highlights that TIBA rescues the short root phenotype of the pct1-2 mutant by acting on a PAT component distinct from the site of action of other PAT inhibitors.

     

Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA^Arg4 as a short-chain peptidyl-tRNA^Arg4 in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA^Arg4 from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on λimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.     

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima’D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.