Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate

The dityrosine layer is a unique structure present in the spore wall of the budding yeast Saccharomyces cerevisiae. The primary constituent of this layer is bisformyl dityrosine. A sporulation-specific protein, Dit1 is localized in the spore cytosol and produces a precursor of bisformyl dityrosine. Although Dit1 is similar to isocyanide synthases, the loss of Dit1 is not rescued by heterologous expression of the Pseudomonas aeruginosa isocyanide synthase, PvcA, indicating that Dit1 does not mediate isocyanidation. The product of Dit1 is most likely formyl tyrosine. Dit1 can produce its product when it is expressed in vegetative cells; however, formyl tyrosine was not detected in the crude cell lysate. We reasoned that formyl tyrosine is unstable and reacts with some molecule to form formyl tyrosine-containing molecules in the cell lysate. In support of this hypothesis, formyl tyrosine was detected when the lysate was hydrolyzed with a mild acid. The same property was also found for bisformyl dityrosine. Bisformyl dityrosine molecules assemble to form the dityrosine layer by an unknown mechanism. Given that bisformyl dityrosine can be released from the spore wall by mild hydrolysis, the process of formyl tyrosine-containing molecule formation may resemble the assembly of the dityrosine layer.

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Accurate MicroRNA Analysis in Crude Cell Lysate by Capillary Electrophoresis-Based Hybridization Assay in Comparison with Quantitative Reverse Transcription-Polymerase Chain Reaction

Analytical Chemistry ◽

10.1021/acs.analchem.7b00707 ◽

2017 ◽

Vol 89 (8) ◽

pp. 4743-4748 ◽

Cited By ~ 13

Author(s):

Liang Hu ◽

Alexander S. Stasheuski ◽

David W. Wegman ◽

Nan Wu ◽

Burton B. Yang ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Capillary Electrophoresis ◽

Reverse Transcription ◽

Hybridization Assay ◽

Chain Reaction ◽

Crude Cell Lysate ◽

Cell Lysate ◽

Polymerase Chain

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A sensitive and robust HPLC method to quantify recombinant antibody fragments in E . coli crude cell lysate

Journal of Chromatography B ◽

10.1016/j.jchromb.2018.02.044 ◽

2018 ◽

Vol 1083 ◽

pp. 242-248 ◽

Cited By ~ 3

Author(s):

Thomas Gundinger ◽

Alexander Pansy ◽

Oliver Spadiut

Keyword(s):

Recombinant Antibody ◽

Hplc Method ◽

Antibody Fragments ◽

Crude Cell Lysate ◽

E Coli ◽

Cell Lysate ◽

Recombinant Antibody Fragments

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Tandem Nanobody: a feasible way to improve the capacity of affinity chromatography

10.22541/au.160690863.36576552/v1 ◽

2020 ◽

Author(s):

Jinheng Fu ◽

Yunxiang Huang ◽

Yinfeng Zhong ◽

Wenyuan Shuai ◽

Hang Zhang ◽

...

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Genetic Manipulation ◽

Binding Capacity ◽

Recombinant Expression ◽

Antigen Recognition ◽

The Self ◽

Crude Cell Lysate ◽

Cell Lysate ◽

Affinity Resin

Nanobodies, referred to the binding domain of the heavy-chain-only antibodies, are the smallest antigen recognition unit. The molecular weight of monomeric nanobodies is about one-tenth of the conventional antibodies. The small size of nanobodies facilitates genetic manipulation and recombinant expression. This study aimed to investigate the effects of nanobody multivalency on the binding capacity of affinity resin. The nanobody (namely AFV), which binds to the fragment crystallizable (Fc) region of immunoglobulin G (IgG), was fused to the N-terminal of HaloTag in the form of monomeric (H-AFV), dimer (H-diAFV), trimer (H-triAFV), and tetramer (H-tetAFV). The fusion proteins were solubly expressed in Escherichia coli yielding at least 9.9 mg L-1. The biolayer interferometry confirmed an increment of avidity as the increase of AFV valences. The four recombinant proteins in crude cell lysate were site-specifically immobilized onto the Halo ligand resin via the self-labeling HaloTag, respectively. The generated affinity resins were able to isolate high purity IgG from mouse plasma. An improvement of 73.7% of the static binding capacity was achieved by the H-diAFV resin as compared to the H-AFV affinity resin.

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