A novel method for selective isotope labeling of bacterially expressed proteins

1995 ◽  
Vol 5 (1) ◽  
pp. 93-96 ◽  
Author(s):  
Karen M. Lee ◽  
Elliot J. Androphy ◽  
James D. Baleja
Keyword(s):  
Isotope Labeling ◽  
Selective Isotope Labeling ◽  
Novel Method

scholarly journals Mass Spectrometric Quantification of the Antimicrobial Peptide Pep19-2.5 with Stable Isotope Labeling and Acidic Hydrolysis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1342
Author(s):  
Sabrina Wohlfart ◽  
Michael Kilian ◽  
Philip Storck ◽  
Thomas Gutsmann ◽  
Klaus Brandenburg ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Isotope Labeling ◽  
Extraction Methods ◽  
Acidic Hydrolysis ◽  
Threatening Condition ◽  
Valuable Procedure ◽  
Life Threatening ◽  
Precise Quantification ◽  
Extraction Properties ◽  
Novel Method

Sepsis is the number one cause of death in intensive care units. This life-threatening condition is caused by bacterial infections and triggered by endotoxins of Gram-negative bacteria that leads to an overreaction of the immune system. The synthetic anti-lipopolysaccharide peptide Pep19-2.5 is a promising candidate for the treatment of sepsis as it binds sepsis-inducing lipopolysaccharides and thus prevents initiation of septic shock. For clinical evaluation precise quantification of the peptide in blood and tissue is required. As the peptide is not extractable from biological samples by commonly used methods there is a need for a new analysis method that does not rely on extraction of the peptide. In order to quantify the peptide by mass spectrometry, the peptide was synthesized containing 13C9,15N1-labeled phenylalanine residues. This modification offers high stability during acidic hydrolysis. Following acidic hydrolysis of the samples, the concentration of 13C9,15N1-labeled phenylalanine determined by LC-MS could be unambiguously correlated to the content of Pep19-2.5. Further experiments validated the accuracy of the data. Moreover, the quantification of Pep19-2.5 in different tissues (as studied in Wistar rats) was shown to provide comparable results to the results obtained with radioactively-labeled (14C) Pep19-2.5- Radioactive labeling is considered as the gold standard for quantification of compounds that refrain from reliable extraction methods. This novel method represents a valuable procedure for the determination of Pep19-2.5 and sticky peptides with unpredictable extraction properties in general.

scholarly journals Selective Isotope Labeling to Facilitate Structural and Dynamics Studies of RNAs by NMR Spectroscopy

2019 ◽  
Vol 116 (3) ◽  
pp. 356a
Author(s):  
Lukasz T. Olenginski ◽  
Owen Becette ◽  
Hyeyeon Nam ◽  
Kehinde M. Taiwo ◽  
Theodore K. Dayie
Keyword(s):  
Nmr Spectroscopy ◽  
Isotope Labeling ◽  
Selective Isotope Labeling

One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

2012 ◽  
Vol 426 (2) ◽  
pp. 126-128 ◽  
Author(s):  
Christopher O’Grady ◽  
Benjamin L. Rempel ◽  
Akosiererem Sokaribo ◽  
Sergiy Nokhrin ◽  
Oleg Y. Dmitriev
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Isotope Labeling ◽  
Selective Isotope Labeling ◽  
One Step

scholarly journals Silkworm Pupae Function as Efficient Producers of Recombinant Glycoproteins with Stable-Isotope Labeling

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1482
Author(s):  
Hirokazu Yagi ◽  
Saeko Yanaka ◽  
Rina Yogo ◽  
Akari Ikeda ◽  
Masayoshi Onitsuka ◽  
...  
Keyword(s):  
Artificial Diet ◽  
Isotope Labeling ◽  
Whole Body ◽  
Enrichment Ratio ◽  
Silkworm Larvae ◽  
Nmr Studies ◽  
Recombinant Glycoproteins ◽  
Silkworm Pupae ◽  
Selective Isotope Labeling ◽  
Bmnpv Bacmid

Baculovirus-infected silkworms are promising bioreactors for producing recombinant glycoproteins, including antibodies. Previously, we developed a method for isotope labeling of glycoproteins for nuclear magnetic resonance (NMR) studies using silkworm larvae reared on an artificial diet containing 15N-labeled yeast crude protein extract. Here, we further develop this method by introducing a technique for the expression of isotope-labeled glycoproteins by silkworm pupae, which has several potential advantages relative to larvae-based techniques in terms of production yield, ease of handling, and storage. Here, we fed fifth instar larvae an artificial diet with an optimized composition containing [methyl-13C]methionine, leading to pupation. Nine-day-old pupae were then injected with recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid for expression of recombinant human immunoglobulin G (IgG). From the whole-body homogenates of pupae, 0.35 mg/pupa of IgG was harvested, which is a yield that is five times higher than can be obtained from larvae. Recombinant IgG, thus prepared, exhibited mainly three kinds of pauci-mannose-type oligosaccharides and had a 13C-enrichment ratio of approximately 80%. This enabled selective observation of NMR signals originating from the methionyl methyl group of IgG, confirming its conformational integrity. These data demonstrate the utility of silkworm pupae as factories for producing recombinant glycoproteins with amino-acid-selective isotope labeling.

Artificially maturated [FeFe] hydrogenase from Chlamydomonas reinhardtii: a HYSCORE and ENDOR study of a non-natural H-cluster

2015 ◽  
Vol 17 (7) ◽  
pp. 5421-5430 ◽  
Author(s):  
Agnieszka Adamska-Venkatesh ◽  
Trevor R. Simmons ◽  
Judith F. Siebel ◽  
Vincent Artero ◽  
Marc Fontecave ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Chlamydomonas Reinhardtii ◽  
Isotope Labeling ◽  
Selective Isotope Labeling ◽  
Insight Into

EPR studies combined with selective isotope labeling provide insight into the electronic structure of the H-cluster in semi-artificial [FeFe] hydrogenase.

A Cost-effective Amino-acid-type Selective Isotope Labeling of Proteins Expressed inLeishmania tarentolae

2009 ◽  
Vol 26 (6) ◽  
pp. 755-761 ◽  
Author(s):  
Silvie Foldynová-Trantírková ◽  
Jana Matulová ◽  
Volker Dötsch ◽  
Frank Löhr ◽  
Ion Cirstea ◽  
...  
Keyword(s):  
Amino Acid ◽  
Isotope Labeling ◽  
Cost Effective ◽  
Amino Acid Type ◽  
Acid Type ◽  
Selective Isotope Labeling
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