Journal:

Article Title: Expression of Recombinant Protein Encoded by LOC387715 in Escherichia coli

doi: 10.1016/j.pep.2007.03.017

Figure Lengend Snippet: A. Schematic diagram of the strategy and process for high and authentical expression of rLOC387715 in E. coli. B. EcoR I + Xho I digestion of pGS21a-LOC387715(ORF)m, showing the expected size of insert (336 bp). C. SDS-PAGE, showing the high expression of the recombinant protein in bacterium BL21(DE3) strain after induction with IPTG (1mM at 37°C for 3 h). The recombinant protein rLOC387715 was about 15% of the bacterial total proteins. Top arrow, the recombinant protein – His-tagged GST-LOC387715 fusion protein that was expressed from pGS21a-LOC387715(ORF)m; bottom arrow, His-tagged GST that was expressed from the vector pGS21a. Std – protein standards.

Article Snippet: LOC387715 cDNA synthesis, cloning and sequencing Self-designed synonymously modified ORF cDNA sequence of LOC387715 – LOC387715(ORF)m was synthesized and cloned in-frame into the bacterial expression vector pGS21a between EcoR I and Xho I sites by GenScript Corporation.

Techniques: Expressing, SDS Page, Recombinant, Plasmid Preparation

Journal:

Article Title: Expression of Recombinant Protein Encoded by LOC387715 in Escherichia coli

doi: 10.1016/j.pep.2007.03.017

Figure Lengend Snippet: A. Detection by anti-GST antibody. B. Detection by anti-6xHis antibody. High expression of the recombinant protein was obtained in bacterium BL21(DE3) strain by the induction of IPTG (1 mM at 37°C for 3 h). Top arrow, the recombinant protein – His-tagged GST-LOC387715 fusion protein that was expressed from pGS21a-LOC387715(ORF)m; bottom arrow, His-tagged GST that was expressed from the vector pGS21a.

Article Snippet: LOC387715 cDNA synthesis, cloning and sequencing Self-designed synonymously modified ORF cDNA sequence of LOC387715 – LOC387715(ORF)m was synthesized and cloned in-frame into the bacterial expression vector pGS21a between EcoR I and Xho I sites by GenScript Corporation.

Techniques: Expressing, Recombinant, Plasmid Preparation

Journal: Journal of Insect Science

Article Title: BmAly Is an Important Factor in Meiotic Progression and Spermatid Differentiation in Bombyx mori (Lepidoptera: Bombycidae)

doi: 10.1093/jisesa/ieu050

Figure Lengend Snippet: Multialignment between the Protein BmAly and its analogs. Homologs amino acids are indicated with a dark grey shadow frame and similar amino acids with light gray shadow. Dark box indicates conserved region 1, dark box with dots indicates conserved region 2, and gray box indicates DIRP domain. Bm, B . mori Aly (GQ999610); Dm, D. melanogaster (AJ277307); Hs, H. sapiens (BC045625); Mm, M . musculus (NM_001103182); Ce, C . elegans (NM_001027844); Nv, N . vitripennis (XM_001605133); Cq, Cu . quinquefasciatus (XM_001862992); Pc, P . humanus corporis (XM_002433084); Dr, D . rerio (XM_002664628); Xt, X . ( Silurana ) tropicalis (XM_002941131); Am, A . mellifera (XM_394339); Tc, T . castaneum (XM_970110).

Article Snippet: The Bmaly PCR product was ligated into vector pMD19-T, and the fragment of Bmaly was excised out with Bam HI/ Xho I and ligated with the expression vector pGS-21a(+) (GenScript, Nanjing, China) to generate the recombinant plasmid pGS-21a(+)- Bmaly .

Techniques:

Journal: Journal of Insect Science

Article Title: BmAly Is an Important Factor in Meiotic Progression and Spermatid Differentiation in Bombyx mori (Lepidoptera: Bombycidae)

doi: 10.1093/jisesa/ieu050

Figure Lengend Snippet: Expression in E. coli and BmAly antibody preparation. (A) SDS-PAGE of BmAly expressed in E. coli . M, protein marker; lane 1, E. coli strain Rosseta transformed with pGS21a(+); lane 2, E. coli strain Rosseta transformed with pGS21a(+)-Bmaly; lane 3, purified recombinant protein. (B) Western blotting for specificity analysis of the BmAly antibody. Left: SDS-PAGE Right: Western blotting. M, protein marker; lanes 1 and 2, E. coli strain BL21 and Rosseta transformed with pGS21a(+)-Bmaly, respectively; lanes 3 and 4, E. coli strain BL21 and Rosseta transformed with pGS21a(+), respectively; Lanes 1′ and 2′, Western blots corresponding to lanes 1 and 2, respectively. The primary antibody was mouse anti-BmAly and the secondary antibody was HRP-conjugated goat antimouse IgG.

Article Snippet: The Bmaly PCR product was ligated into vector pMD19-T, and the fragment of Bmaly was excised out with Bam HI/ Xho I and ligated with the expression vector pGS-21a(+) (GenScript, Nanjing, China) to generate the recombinant plasmid pGS-21a(+)- Bmaly .

Techniques: Expressing, SDS Page, Marker, Transformation Assay, Purification, Recombinant, Western Blot

Journal: Journal of Insect Science

Article Title: BmAly Is an Important Factor in Meiotic Progression and Spermatid Differentiation in Bombyx mori (Lepidoptera: Bombycidae)

doi: 10.1093/jisesa/ieu050

Figure Lengend Snippet: Subcellular localization of BmAly. BmN cells were treated with anti-BmAly antibody, followed by treatment with FITC-conjugated goat antimouse IgG, and the nucleus was treated with DAPI (blue), which were examined under Inverted fluorescence microscope. From left to right, green fluorescence for FITC-treated BmAly, DAPI-treated nucleus. For the control, preimmune serum was used as the primary antibody. Original magnification for BmN cells =200×.

Article Snippet: The Bmaly PCR product was ligated into vector pMD19-T, and the fragment of Bmaly was excised out with Bam HI/ Xho I and ligated with the expression vector pGS-21a(+) (GenScript, Nanjing, China) to generate the recombinant plasmid pGS-21a(+)- Bmaly .

Techniques: Fluorescence, Microscopy, Control

Journal: Journal of Insect Science

Article Title: BmAly Is an Important Factor in Meiotic Progression and Spermatid Differentiation in Bombyx mori (Lepidoptera: Bombycidae)

doi: 10.1093/jisesa/ieu050

Figure Lengend Snippet: Bmaly expression. Q-PCR analysis of Bmaly mRNA at different organisms, different stages of testis development. Testes, ovaries, silk gland, malpighian tubule, fat body, and midgut were dissected from silkworms at the times indicated, and cDNA was synthesized as described above.

Article Snippet: The Bmaly PCR product was ligated into vector pMD19-T, and the fragment of Bmaly was excised out with Bam HI/ Xho I and ligated with the expression vector pGS-21a(+) (GenScript, Nanjing, China) to generate the recombinant plasmid pGS-21a(+)- Bmaly .

Techniques: Expressing, Synthesized

Journal: Journal of Insect Science

Article Title: BmAly Is an Important Factor in Meiotic Progression and Spermatid Differentiation in Bombyx mori (Lepidoptera: Bombycidae)

doi: 10.1093/jisesa/ieu050

Figure Lengend Snippet: Development of sperm cells. (A) Testis of the first day of fifth instar larvae. (B) Testis of the first day of fifth instar larvae, which was injected with Bmaly siRNA-657 at third and fourth instars. ca, cellula apicalis; ps, primary spermatocytes; ss, secondary spermatocytes; pr, prespermatid. (C) A schematic showing the progression of spermatogenesis in B. mori. Spermatogonial cell born at the apical tip surround the teloblast, they undergo six rounds of synchronous mitotic divisions to produce cysts of 64 primary spermatocysts, then they go to larger secondary spermatocytes, followed by sperm bundle which containing mature sperm. (D) The level of Bmaly siRNA-657 mRNA-injected silkworms compared with that in normal (no injection), DEPC H 2 O, and Bmaly siRNA-96, respectively.

Article Snippet: The Bmaly PCR product was ligated into vector pMD19-T, and the fragment of Bmaly was excised out with Bam HI/ Xho I and ligated with the expression vector pGS-21a(+) (GenScript, Nanjing, China) to generate the recombinant plasmid pGS-21a(+)- Bmaly .

Techniques: Injection

Journal: PLoS ONE

Article Title: Structural and Functional Characterization of DUF1471 Domains of Salmonella Proteins SrfN, YdgH/SssB, and YahO

doi: 10.1371/journal.pone.0101787

Figure Lengend Snippet: A : Multiple sequence alignment of DUF1471 paralogues from S. Typhimurium, as well as E. coli YbiM, for which there is no close homolog in Salmonella . Alignment of SrfN, YahO, SssB-I and SssB-III is structure-based over the entire structured sequence (SrfN residues 22–91), other alignments are sequence-based and are between core regions only (SrfN residues 35–91) because sequence identity to SrfN residues 22–38 is low and alignments in this region are uncertain. Secondary structure in SrfN, YahO, SssB-I, and SssB-III is indicated above: E = extended (β-sheet) structure, H = helix. The core residues of the sulfate-binding motif in SrfN are indicated with asterisks. Conserved sequence motifs identified by Rudd are underlined. Other conserved residues are highlighted in green or dark grey. Two notable loop regions in the structure are also indicated. SrfN and YahO both have C-terminal tag sequences LEHHHHHH that are not shown. Light grey highlighted portions indicate likely signal sequences for periplasmic localization that are known or likely to be cleaved by a signal peptidase. In the case of SrfN and YahO, the signal sequence was proven experimentally to be cleaved during heterologous expression in E. coli . Inter-domain regions of SssB are not shown. Lower case letters in SssB-III (C-terminal domain) indicate residues with missing electron density in the X-ray structure. Highly conserved residues are indicated by highlighting (blue = hydrophobic, green = polar), somewhat conserved residues are indicated with grey highlighting. The following sequences are listed ( S. Typhimurium LT2 locus and UniProt/TrEMBL numbers in parentheses): SrfN (STM0082/Q7CR88), YjfY (STM4389/Q8ZK84), YhcN (STM3361/Q8ZLP6), YcfR seq. I (STM1214/Q8ZQ03), YcfR seq. II (STM3362/Q7CPN0), YahO (STM0366/Q7CR49), YbiJ (STM0823/Q7CQW3), YkgI (STM0565/Q7CR04), YjfO (STM4379/Q8ZK92), YjfN (STM4378/Q8ZK93), SssB (STM1478/Q8ZPL1), YbiM/McbA ( E. coli , P0AAX6). B : Unrooted phylogenetic tree (phylogram) constructed from ten diverse genera from the Enterobacteriaceae. Major branches containing Salmonella and E. coli subfamily members are indicated. C : Multiple sequence alignment of SrfN homologues: a subfamily of DUF1471 proteins. For each sequence and abbreviated organism name listed, the full genus and species name, protein/ORF name, database accession number, and similarity to SrfN, excluding the signal sequence, are as follows: Sty, Salmonella enterica Typhimurium, STM0082 (SrfN), NP_459087 and many other Salmonella strains; Sbo, Salmonella bongori , SBG_0068, YP_004728986 (93%); Cro, Citrobacter rodentium , ROD_12311, YP_003364817 (80%); Eho, Enterobacter hormaechei , HMPREF9086_0329, ZP_08496071 (65%); Eae, Enterobacter aerogenes EAE_13230, YP_004592839 (70%); Kpn, Klebsiella pneumonia , KPK_4095, YP_002239898 (68%); Pan, Pantoea sp., Pat9b_3745, YP_004117591 (61%). Notes: Other Salmonella, Klebsiella, and Enterobacter species and strains contain identical or nearly identical sequences to the representatives shown here. However, some Pantoea species do not contain homologues that fall within this DUF1471 subfamily.

Article Snippet: To prepare samples of SrfN for characterization by mass spectrometry, confirmation and refinement of the original NMR structure, and characterization of binding interactions, the recombinant full length STM0082 (SrfN) gene was synthesized by GenScript (Piscataway, NJ) and incorporated into the pGS-21a expression vector encoding a C-terminal 6X-His tag.

Techniques: Sequencing, Binding Assay, Expressing, Construct