Gene expression is regulated by chromatin DNA methylation and other features, including histone post-translational modifications (PTMs), chromatin remodelers and transcription factor occupancy. A complete understanding of gene regulation will require the mapping of these chromatin features in small cell number samples. Here we describe a novel genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq). NEED-seq offers antibody-targeted controlled nicking by Nt.CviPII-pGL fusion to study specific protein-DNA complexes in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide profile of chromatin-associated proteins and histone PTMs. Additionally, NEED-seq of lamin B1 and B2 demonstrated their association with heterochromatin. Lamin B1- and B2-associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1 and B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1 and B2 partitioning to facultative and constitutive heterochromatin, respectively, and were associated with neuronal development. Thus, lamin B1 and B2 show structural and functional partitioning in mammalian nucleus.

A genome-wide chromatin profiling technology, named as Nicking Enzyme Epitope targeted DNA sequencing (NEED-seq) in which antibody-targeted controlled nicking by Nt.CviPII-pGL is used to study specific protein-DNA complexes. NEED-seq is performed in situ in formaldehyde fixed cells, allowing for both visual and genomic resolution of epitope bound chromatin. When applied to nuclei, NEED-seq yielded genome-wide chromatin associated proteins and histone post-translational modifications (PTMs). NEED-seq of lamin B1/B2 demonstrated their association with heterochromatin. Lamin B1 and B2 associated domains (LAD) segregated to three different states, and states with stronger LAD correlated with heterochromatic marks. Hi-C analysis displayed A and B compartment with equal lamin B1/B2 distribution, although methylated DNA remained high in B compartment. LAD clustering with Hi-C resulted in subcompartments, with lamin B1-B2 partitioning to facultative and constitutive heterochromatin respectively and were associated with neuronal development. Thus, lamin B1 and B2 have structural and functional partitioning in mammalian nucleus. ![Figure 1:][1] Figure 1: Graphical abstract: Model depicting association of lamin B1 and B2 in A (facultative heterochromatin) and B (constitutive heterochromatin) compartmentalization. ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

N-glycosylation is implicated in cancers and aberrant N-glycosylation is recognized as a hallmark of cancer. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 displays hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells. This comparative site-specific N-glycoproteome analysis provides a pool of potential N-glycosylation-related cancer biomarkers, but also gives insights into the M6P pathway in cancer.

High confidence methods are needed for determining the glycosylation profiles of complex biological samples as well as recombinant therapeutic proteins. A common glycan analysis workflow involves liberation of N-glycans from glycoproteins with PNGase F or O-glycans by hydrazinolysis prior to their analysis. This method is limited in that it does not permit determination of glycan attachment sites. Alternative proteomics-based workflows are emerging that utilize site-specific proteolysis to generate peptide mixtures followed by selective enrichment strategies to isolate glycopeptides. Methods designed for the analysis of complex samples can yield a comprehensive snapshot of individual glycans species, the site of attachment of each individual glycan and the identity of the respective protein in many cases. This chapter will highlight advancements in enzymes that digest glycoproteins into distinct fragments and new strategies to enrich specific glycopeptides.

N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.

Cell-free protein synthesis is an attractive method for generating enzyme/protein variants for simplified functional analysis as both in vitro protein expression and analysis may often be performed in a single vial or well. Today, researchers may choose from multiple commercial cell lysate products or reconstituted systems which are compatible with either mRNA, linear DNA or plasmid DNA templates. Here we provide guidance for optimal design of the genetic elements within linear and plasmid DNA templates which are required to reliably practice cell-free protein synthesis. Protocols are presented for generating linear DNA templates, and data are presented to show that linear DNA templates may in many cases provide robust protein yields even when employing an Escherichia coli lysate for protein synthesis. Finally, the use of linear DNA templates makes it possible to bypass all cell cultivation steps and proceed from PCR amplification of synthetic DNA to generation of target protein in a matter of hours.

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EM-seq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity.

Genetic Engineering & Biotechnology NewsVol. 36, No. 5 Bioprocessing TutorialPurifying Recombinant His-Tagged ProteinsImproving the Process with Use of Genetically Tailored Expression Host NiCo21(DE3)James C. SamuelsonJames C. SamuelsonJames C. Samuelson, Ph.D. (E-mail Address: samuelson@neb.com), is a senior scientist within the protein expression and modification division at New England Biolabs. Website: www.neb.com.Search for more papers by this authorPublished Online:26 Feb 2016https://doi.org/10.1089/gen.36.05.15AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 36Issue 5Mar 2016 Information© 2016 by GEN PublishingTo cite this article:James C. Samuelson.Purifying Recombinant His-Tagged Proteins.Genetic Engineering & Biotechnology News.Mar 2016.26-27.http://doi.org/10.1089/gen.36.05.15Published in Volume: 36 Issue 5: February 26, 2016PDF download

The requirements for target protein folding in M13 phage display are largely underappreciated. Here we chose Fbs1, a carbohydrate binding protein, as a model to address this issue. Importantly, folding of Fbs1 is impaired in an oxidative environment. Fbs1 can be displayed on M13 phage using the SRP or Sec pathway. However, the displayed Fbs1 protein is properly folded only when Fbs1 is translocated via the SRP pathway and displayed using Escherichia coli cells with a DsbA-negative periplasm. This study indicates M13 phage display may be improved using a system specifically designed according to the folding requirements of each target protein.

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

1. A Tripartite Fusion System for the Selection of Protein Variants with Increased Stability in vivo Linda Foit and James Bardwell 2. Determining Enzyme Kinetics via Isothermal Titration Calorimetry Neil A. Demarse, Marie C. Killian, Lee D. Hansen, and Colette F. Quinn 3. GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria Olga Paley, Giulia Agnello, Jason Cantor, Tae Hyun Yoo, George Georgiou, and Everett Stone 4. Flow Cytometric Assays for Interrogating LAGLIDADG Homing Endonuclease DNA Binding and Cleavage Properties Sarah K. Baxter, Abigail R. Lambert, Andrew M. Scharenberg, and Jordan Jarjour 5. TAL Effector Nuclease (TALEN) Engineering Ting Li and Bing Yang 6. In vitro Evolution of Enzymes Misha V. Golynskiy, John C. Haugner III, Aleardo Morelli, Dana Morrone, and Burckhard Seelig 7. Residue-Specific Incorporation of Unnatural Amino Acids into Proteins In vitro and In vivo Amrita Signh-Blom, Randall A. Hughes, and Andrew. D. Ellington 8. Reconstructing Evolutionary Adaptive Paths for Protein Engineering Megan F. Cole, Vanessa E. Cox, Kelsey L. Gratton, and Eric A. Gaucher 9. Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria Bryan M. Swingle 10. FX Cloning: A Versatile High-Throughput Cloning System for Characterization of Enzyme Variants Eric R. Geertsma 11. Use of Sulfolobus solfataricus PCNA Subunit Proteins to Direct the Assembly of Multimeric Enzyme Complexes Hidehiko Hirakawa and Teruyuki Nagamune 12. Gene Synthesis by Assembly of Deoxyuridine Containing Oligonucleotides Romualdas Vaisvila and Jurate Bitinaite 13. Protein Engineering: Single or Multiple Site-Directed Mutagenesis Pei-Chung Hsieh and Romualdas Vaisvila 14. Gene Assembly and Combinatorial Libraries in S. cerevisiae via Reiterative Recombination Nili Ostrov, Laura M. Wingler, and Virginia Cornish 15. Promiscuity-Based Enzyme Selection for Rational Directed Evolution Experiments Sandeep Chakraborty, Renu Minda, Lipika Salaye, Abhaya M. dandekar, Swapan K. Bhattacharjee, and Basuthkar J. Rao 16. Rational Protein Sequence Diversification by Multi-Codon Scanning Mutagenesis Jia Liu and T. Ashton Cropp 17. Screening Libraries for Improved Solubility: Using E. coli Dihydrofolate Reductase as a Reporter Jian-Wei Liu and David Ollis 18. In Vitro Directed Evolution of Enzymes Expressed by E. coli in Micro-Titre Plates Bradley J. Stevenson, Sylvia H.C. Yip, and David L. Ollis

Genetic Engineering & Biotechnology NewsVol. 32, No. 3 TutorialDisulfide-Bonded Protein Production in E. coliInvolvement of Disulfide Bond Isomerase Improves Protein FoldingPublished Online:1 Feb 2012https://doi.org/10.1089/gen.32.3.17AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byProduction of antibodies in SHuffle Escherichia coli strainsStrategies for recombinant production of antimicrobial peptides with pharmacological potential16 June 2020 | Expert Review of Clinical Pharmacology, Vol. 13, No. 4High cell density cultivation of Δgor/ΔtrxB E. coli in a chemically defined minimal medium with an enhanced iron concentrationProcess Biochemistry, Vol. 73Use of the SHuffle Strains in Production of Proteins1 August 2016 | Current Protocols in Protein Science, Vol. 85, No. 1Production of Disulfide‐Bonded Proteins in Escherichia coli1 October 2014 | Current Protocols in Molecular Biology, Vol. 108, No. 1 Volume 32Issue 3Feb 2012 Information© 2012 by GEN PublishingTo cite this article:James C. Samuelson, Thomas B. Causey, Mehmet Berkmen.Disulfide-Bonded Protein Production in E. coli.Genetic Engineering & Biotechnology News.Feb 2012.35-35.http://doi.org/10.1089/gen.32.3.17Published in Volume: 32 Issue 3: February 1, 2012PDF download

EcoP15I is the prototype of the Type III restriction enzyme family, composed of two modification (Mod) subunits to which two (or one) restriction (Res) subunits are then added. The Mod subunits are responsible for DNA recognition and methylation, while the Res subunits are responsible for ATP hydrolysis and cleavage. Despite extensive biochemical and genetic studies, there is still no structural information on Type III restriction enzymes. We present here small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analysis of the EcoP15I holoenzyme and the Mod(2) subcomplex. We show that the Mod(2) subcomplex has a relatively compact shape with a radius of gyration (R(G)) of ∼37.4 Å and a maximal dimension of ∼110 Å. The holoenzyme adopts an elongated crescent shape with an R(G) of ∼65.3 Å and a maximal dimension of ∼218 Å. From reconstructed SAXS envelopes, we postulate that Mod(2) is likely docked in the middle of the holoenzyme with a Res subunit at each end. We discuss the implications of our model for EcoP15I action, whereby the Res subunits may come together and form a "sliding clamp" around the DNA.

ABSTRACT Recombinant His-tagged proteins expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted 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 flowthrough 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.

Escherichia coli is a versatile and popular tool for heterologous protein production. Some of the reasons for its popularity include rapid growth, a variety of portable vectors, relatively simple genetics, and the potential for high-density cultivation. In addition, the extensive laboratory use of E. coli has resulted in technologies to target protein overexpression to various intracellular compartments. This is advantageous because these compartments have different environments that may facilitate folding of particular proteins of interest. This chapter discusses the properties of many of the E. coli strains available for protein expression in order to facilitate the choice of the best expression host for a particular protein of interest.