scholarly journals Type III CRISPR complexes from Thermus thermophilus.

2016 ◽  
Vol 63 (2) ◽  
Author(s):  
Marta Szychowska ◽  
Wojciech Siwek ◽  
Damian Pawolski ◽  
Asgar Abbas Kazrani ◽  
Krzysztof Pyrc ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Mass Spectroscopy ◽  
Thermus Thermophilus ◽  
Size Exclusion ◽  
Acquired Immunity ◽  
Type I ◽  
Type Iii ◽  
Exclusion Chromatography

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.

scholarly journals Bifidobacterium longum Endogalactanase Liberates Galactotriose from Type I Galactans

2005 ◽  
Vol 71 (9) ◽  
pp. 5501-5510 ◽  
Author(s):  
Sandra W. A. Hinz ◽  
Marieke I. Pastink ◽  
Lambertus A. M. van den Broek ◽  
Jean-Paul Vincken ◽  
Alphons G. J. Voragen
Keyword(s):  
Molecular Mass ◽  
Transmembrane Domain ◽  
Bifidobacterium Longum ◽  
Cell Extract ◽  
Size Exclusion ◽  
Glycoside Hydrolase Family ◽  
Type I ◽  
Native Molecular Mass ◽  
Exclusion Chromatography ◽  
Hydrolase Family

ABSTRACT A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(−) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37°C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The Km and V max for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (β1→4)galactans and (β1→4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (β1→3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

Molecular mass determinations in coal-derived liquids by MALDI mass spectrometry and size-exclusion chromatography

Fuel ◽  
1997 ◽  
Vol 76 (13) ◽  
pp. 1225-1233 ◽  
Author(s):  
Maria-Jesus Lázaro ◽  
Alan A. Herod ◽  
Mike Cocksedge ◽  
Mark Domin ◽  
Rafael Kandiyoti
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Maldi Mass Spectrometry ◽  
Size Exclusion ◽  
Exclusion Chromatography

Oat spelts xylan molecular mass estimation by size exclusion chromatography

2003 ◽  
Vol 53 (3) ◽  
pp. 297-304 ◽  
Author(s):  
Andrei Sarbu ◽  
Fernando Gonçalves ◽  
Maria Norberta de Pinho
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Size Exclusion ◽  
Mass Estimation ◽  
Exclusion Chromatography

scholarly journals Immunoassay for Sex Hormone-Binding Globulin in Undiluted Serum Is Influenced by High-Molecular-Mass Aggregates

2005 ◽  
Vol 51 (2) ◽  
pp. 401-407 ◽  
Author(s):  
Markus Thaler ◽  
Jochen Metzger ◽  
Anita Schreiegg ◽  
Barbara Denk ◽  
Andreas Gleixner ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
High Molecular Mass ◽  
Sex Hormone ◽  
Size Exclusion ◽  
Sex Hormone Binding Globulin ◽  
Hormone Binding ◽  
Spr Biosensor ◽  
Undiluted Serum ◽  
Exclusion Chromatography

Abstract Background: The new Elecsys® chemiluminescence assay for measurement of homodimeric sex hormone-binding globulin (SHBG) was designed for use with undiluted serum, in contrast to other methods that require predilution. During assay development, unexpected calibration difficulties were observed that were attributable to particular biochemical properties of the highly concentrated SHBG in solution. Methods: We used a surface plasmon resonance (SPR) biosensor, which enables biomolecular interaction analysis of SHBG, and size-exclusion chromatography for this investigation. The immunoassay was evaluated for imprecision, linearity, and suitability of the dilution medium, and the method was compared with an IRMA for SHBG. Results: The SPR biosensor characterized the special protein properties of SHBG in various concentrations. Above 200 nmol/L there was a strong tendency toward formation of high-molecular-mass aggregates. This was also detectable by size-exclusion chromatography and could be reversed by simple dilution of the sample. On the basis of these results, the dynamic measuring range of the SHBG assay is restricted to 0.350–200 nmol/L. Assay evaluation on a 2010 analyzer revealed excellent precision (CV ≤2.5%). Mean recoveries were 84.2–98.8%. Intermethod comparison with an IRMA yielded a satisfactory concordance of the two assays with a Spearman correlation coefficient of 0.8807. Conclusions: Aggregates of human SHBG may have a detrimental impact on the accurate measurement of the protein if measurements are performed with undiluted serum samples. Further work is needed to clarify whether these high-molecular-mass aggregates influence the free fraction of steroid hormones in vivo.

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