220 research outputs found
Co-expression of lupanine hydroxylase and pyrroloquinoline quinone 2 leads to assembled and active recombinant lupanine hydroxylase in the 3 Escherichia coli periplasm
Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy
Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b 5 (CYT b 5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb 5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb 5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS-PAGE and western blot analysis
Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing
Functional genomics via metabolic footprinting: monitoring metabolite secretion by Escherichia coli tryptophan metabolism mutants using FT-IR and direct injection electrospray mass spectrometry
We sought to test the hypothesis that mutant bacterial strains could be discriminated from each other on the basis of the metabolites they secrete into the medium (their āmetabolic footprintā), using two methods of āglobalā metabolite analysis (FTāIR and direct injection electrospray mass spectrometry). The biological system used was based on a published study of Escherichia coli tryptophan mutants that had been analysed and discriminated by Yanofsky and colleagues using transcriptome analysis. Wild-type strains supplemented with tryptophan or analogues could be discriminated from controls using FTāIR of 24 h broths, as could each of the mutant strains in both minimal and supplemented media. Direct injection electrospray mass spectrometry with unit mass resolution could also be used to discriminate the strains from each other, and had the advantage that the discrimination required the use of just two or three masses in each case. These were determined via a genetic algorithm. Both methods are rapid, reagentless, reproducible and cheap, and might beneficially be extended to the analysis of gene knockout libraries
Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis
Sheep pancreatic microsomes as an alternative to the dog source for studying protein translocation
Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts
Isolated outer envelope membrane from pea (Pisum
sativum L.) chloroplasts can be used in vitro to study
binding and partial translocation of precursor proteins
destined for the inside of the organelle. Efficient binding
to a receptor protein on the outside of the membrane
vesicle and generation of a translocation intermediate
depends strictly on the presence of ATP. Protease
treatment of the translocation intermediate demonstrates
its insertion into the membrane. The membraneinserted
precursor protein cannot be extracted by 1 M
NaCl and is also NaOH resistant to a large extent. Mild
solubilization of outer envelope membranes by detergent
resulted in the isolation of a complex which still
contained the precursor protein. We have identified a
constitutively expressed homologue hsc 70 as part of
this membrane complex. Antibodies against hsp 70
(inducible heat shock protein 70) were able to immunoprecipitate
the complex bound precursor protein.
A second protein of 86 kDa molecular weight (OEP 86)
from the outer envelope membrane was also identified
as a major component of this complex
A reassessment of the assay for the asialoglycoprotein receptor and its use in the quantification of receptor distribution in hepatocytes
Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane
Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST
Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14)
- ā¦