NEB Scientific Posters

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1. Development of a Rapid Method for Genome Engineering in a Quest Towards Customized Protein Expression Strains (2015)

   The ability to produce high levels of recombinant protein has become one of the cornerstones of biological sciences. Purified proteins allow us to obtain information about their specificities and even structures. To date, many proteins have been expressed and purified from engineered host cells, such as E. coli, due to the relative simplicity of the process. Unfortunately, many other proteins of interest are not readily expressed or expressed as insoluble aggregates in bacterial hosts. Many advances have been made over the past several decades to understand and improve folding and solubility of recombinant proteins within their new hosts, however, there are still many enzymes that do not fold well.
   
   Each protein is unique and we believe that creating customized expression strains for each difficult protein is a strategy that is currently under-utilized, in part because of the time and effort required for extensive genome modification in E. coli. Here, we present the development of a novel, rapid method for E. coli genome engineering, allowing us to make markerless genome modifications in 48 hours. As an example of such modification, we restored wild-type lon allele in a B strain and demonstrate its beneficial effect on production of a membrane protein.

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2. miRNA Detection by Ligation and Amplification of Complementary DNA Oligos Using SPlintR® Ligase (2015)

   Ligation of two adjacent DNA oligonucleotides splinted by RNA has historically been difficult to achieve. We have discovered that SplintR® ligase (Chlorella virus DNA ligase) is much more efficient in this ligation than T4 DNA Ligase, which was traditionally used for this application. When these ligases were compared using the same RNA:DNA substrates the SplintR^® enzyme achieved complete ligation with 10X less ligase and was 15X faster than T4 DNA Ligase [1].

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3. Improving Sample Workflow: Rapid PNGase F for Accurate Antibody Characterization (2015)

   The characterization of glycoprotein structure is becoming increasingly sophisticated, as regulatory agencies require multiple attributes to be measured during development, production, and formulation of biological drugs. Precise determination of N- and O-glycosylation, site occupancy, disulfide shuffling, misassembly, deamidation, oxidation, etc, require robust methods for sample preparation, to facilitate mass spectrometry analysis. Enzymes for glycan removal, along with specific proteases, are critical to these studies. Improved methods where glycosidases are combined, and/or coupled with labeling reactions or protease digestion, maximize reproducibility by eliminating handling errors. These methods, in turn, permit a more stringent definition of an original, biosimilar, or biobetter, facilitating formulation and process development innovations. We present in this poster new glycan removal protocols, including fast deglycosylation (using Rapid PNGase F) and deglycosylation of intact plant-derived glycoproteins (using PNGase Ar). These reactions were coupled with a simplified and versatile glycan labeling reaction by reductive amination, suitable for glycans lacking a glycosylamine end group. Also, glycosidase combinations were tested for complete N- and O-glycan removal, to facilitate proteomic analysis for glycoproteins that are heavily glycosylated. Finally, an enzyme mix containing PNGase F and Trypsin was used to prepare an IgG sample for peptide mapping. This abbreviated workflow maintained sensitivity and reproducibility

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4. In Vitro Reconstitution of Thermococcus Species 9°N Okazaki Fragment Maturation (2015)

   During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications and generation of doublestranded DNA breaks.To address the components required for the process, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.

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5. Small RNA-Mediated DNA Methyltransferase 1 Inhibition Causes DNA Methylation Alteration in Mammalian Cells (2015)

   Uses hemimethylated DNA as substrate.
   

   • Responsible for CpG methylation maintenance.
   • Dysregulation of DNMT1 causes aberrant methylation in cancer.
   • Highly abundant transcripts inhibit DNMT1 activity in vivo (asCEBPA).
   • LncRNA (Kcnq1ot1) recruits DNMT1 to imprinted genes and somatic DMR.

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6. NiCo21(DE3): A BL21(DE3) Derivative Designed for Expression and Purification of His-tagged Recombinant Protein (2015)

   Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly co-eluted with native E. coli proteins, especially if the recombinant protein is expressed at a low level. The E. coli contaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineered E. coli BL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA and optionally AceE fused at their C-terminus to a chitin binding domain (CBD), and the protein GlmS with six surface histidines replaced by alanines. We show that each E. coli CBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flow-through step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

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7. Crystal Structure of the 8 bp-Specific Restriction Enzyme SwaI (2015)

   SwaI, a Type IIP restriction enzyme from Staphylococcus warneri cleaves the symmetric sequence ATTT|AAAT, producing fragments with blunt ends (‘|’ = cleavage site). We solved the crystal structure of SwaI alone, of SwaI bound to uncleaved DNA in the presence of Ca²+ ions, and of SwaI bound to cleaved DNA in the presence of Mg²+ ions. We describe these structures, and compare them to that of PacI, which cleaves the related 8-bp sequence, TTAAT|TAA.

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8. One Ring To Bind Them All: Is HemeBiosynthesis A Factor In Wolbachia-Filarial Nematode Endosymbiosis? (2015)

   Transmitted by insect vectors, human nematode-based filariasis causes debilitating diseases affecting nearly 150 million people with 1.2 billion individuals at risk in 80 countries. Genomic sequencing has revealed that many human filarial nematodes, such as Wuchereria bancrofti, Brugia malayi and B. timori, (causative agents of lymphatic filariasis (LF)) and Onchocerca volvulus (causative agent of onchocerciasis (river blindness)) contain the obligate endosymbiont, Wolbachia. Genome sequencing of B. malayi (Bm) and its Wolbachia (wBm) identified a number of metabolites implicated in the host-endosymbiont interaction, one of which was heme, a co-factor in a number of enzymes and essential to many biological processes. Although the Bm genome encodes a functional ferrochelatase gene (the final step in the heme biosynthetic pathway and a product of lateral gene transfer), like other nematodes, it is incapable of synthesizing heme. However, the wBm genome contains a functional heme synthesis pathway, leading to the hypothesis that wBm may supply Bm with heme

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9. Molecular Diagnostics for Gastrointestinal Parasites and Impact on Intestinal Microbiota in Rural Agentinian Children (2015)

   • 2 billion GI parasite infections worldwide
     • Poorest and resource-deprived communities
   • Standard method of diagnosis: Stool microscopy
     • Sensitivity variable depending on prevalence, species, and concentration method
       • 50-90% sensitivity
     • Underestimates polyparasitism
   • qPCR is rapid, quantitative, and high throughput species-specific method
   • GI parasites may disrupt normal intestinal microbiota
     • Decreased biodiversity is associated with disease
       • Malabsorption
       • Inflammatory bowel diseases

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