cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase

A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined. The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli. The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active. The recombinant SHMT was purified to homogeneity with a yield of 10 mg/1 bacterial culture. The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme. The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme. The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5′-phosphate as an internal aldimine. The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5′-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT. This overexpression of eukaryotic tetrameric SHMT in E. coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme

MoS₂-ZnO Nanocomposite Mediated Immunosensor for Non-Invasive Electrochemical Detection of IL8 Oral Tumor Biomarker

In order to support biomolecule attachment, an effective electrochemical transducer matrix for biosensing devices needs to have many specialized properties, including quick electron transfer, stability, high surface area, biocompatibility, and the presence of particular functional groups. Enzyme-linked immunosorbent assays, gel electrophoresis, mass spectrometry, fluorescence spectroscopy, and surface-enhanced Raman spectroscopy are common techniques used to assess biomarkers. Even though these techniques provide precise and trustworthy results, they cannot replace clinical applications because of factors such as detection time, sample amount, sensitivity, equipment expense, and the need for highly skilled individuals. For the very sensitive and targeted electrochemical detection of the salivary oral cancer biomarker IL8, we have created a flower-structured molybdenum disulfide-decorated zinc oxide composite on GCE (interleu-kin-8). This immunosensor shows very fast detection; the limit of detection (LOD) for interleukin-8 (IL8) detection in a 0.1 M phosphate buffer solution (PBS) was discovered to be 11.6 fM, while the MoS2/ZnO nanocomposite modified glassy carbon electrode (GCE) demonstrated a high catalytic current linearly from 500 pg to 4500 pg mL−1 interleukin-8 (IL8). Therefore, the proposed biosensor exhibits excellent stability, high accuracy sensitivity, repeatability, and reproducibility and shows the acceptable fabrication of the electrochemical biosensors to detect the ACh in real sample analysis

High level constitutive expression of luciferase reporter by lsd90 promoter in fission yeast.

Because of a large number of molecular similarities with higher eukaryotes, the fission yeast Schizosaccharomyces pombe has been considered a potentially ideal host for expressing human proteins having therapeutic and pharmaceutical applications. However, efforts in this direction are hampered by lack of a strong promoter. Here, we report the isolation and characterization of a strong, constitutive promoter from S. pombe. A new expression vector was constructed by cloning the putative promoter region of the lsd90 gene (earlier reported to be strongly induced by heat stress) into a previously reported high copy number vector pJH5, which contained an ARS element corresponding to the mat2P flanking region and a truncated URA3m selectable marker. The resulting vector was used to study and compare the level of expression of the luciferase reporter with that achieved with the known vectors containing regulatable promoter nmt1 and the strong constitutive promoter adh1 in S. pombe and the methanol-inducible AOX1 promoter in Pichia pastoris. Following growth in standard media the new vector containing the putative lsd90 promoter provided constitutive expression of luciferase, at a level, which was 19-, 39- and 10-fold higher than that achieved with nmt1, adh1 and AOX1 promoters, respectively. These results indicate a great potential of the new lsd90 promoter-based vector for commercial scale expression of therapeutic proteins in S. pombe

Constitutive expression of the Firefly luciferase (<i>Fluc</i>) under lsd90 promoter.

<p>Cultures of strain <i>SPJ25</i> harboring the vector constructs pJH6c-Fluc and pJH6c were grown in selective media (PMA ura⁻) at 30°C and 200 rpm. Aliquots were taken at the indicated time points and subjected to protein extraction. (A) Luciferase activity was determined using the Luciferase Assay System (Promega, USA). Assays were done in triplicate and the average luminescence values were plotted against the indicated time points. (B) Graph showing growth kinetics of cultures up to early stationary phase. (C) The histogram shows the cell density in Cells/ml at the indicated time points of culture.</p

Relative-qPCR analysis of Luciferase mRNA.

<p>RNA isolated form cells harvested at time points showing maximum level of Fluc RNA under the control of different vectors was subjected to real time RTPCR analysis. Samples were analyzed in triplicate and displayed as histogram. The relative Fluc/<i>act1</i> RNA levels expressed by different vectors are displayed after normalization with respect to the vector pJH6c-Fluc. The time points were: pJH6c-Fluc: 32 hrs; pART1-Fluc: 48 hrs; pREP3X-Fluc: 16 hrs, pPIC3.5-Fluc: 96 hrs.</p

Time course of Fluc-expression under control of the <i>nmt1</i> promoter of <i>S. pombe</i>.

<p>Cultures of strain <i>SPJ25</i> harboring the vector constructs pREP3X-Fluc and pREP3X were grown in selective media (PMA leu⁻) at 30°C and 200 rpm. Initially the cultures were grown in medium containing thiamine and then sub-cultured in medium lacking thiamine for the indicated time points. (A) Luciferase activity measured in RLU and (B) growth kinetics of cultures.</p

<b>P</b>rimers used in this study.

<p><b>P</b>rimers used in this study.</p

Comparison of levels of expression of luciferase using different expression systems.

<p>Comparison of levels of expression of luciferase using different expression systems.</p

Time course of luciferase expression under control of the <i>AOX1</i> promoter of <i>P. pastoris</i>.

<p>Cultures of recombinant strain <i>GS115</i> harboring the <i>Fluc</i>-containing expression vector pPIC3.5-Fluc and the control vector pPIC3.5 were grown in suitable medium up to six days. Luciferase assay was performed in triplicate and activity was plotted against indicated time points.</p

Schematic diagram of the Fluc-expression vectors used in this study.

<p>Restriction map of Fluc-expression vectors; (A) pJH6c-Fluc, (B) pART1-Fluc, (C) pREP3X-Fluc and (D) pPIC3.5-Fluc. Approximately 1.0 kb upstream region of gene <i>SPAC1F8.02C/lsd90</i> of <i>S. pombe</i> was PCR amplified and inserted into the plasmid pJH5 at SphI/NdeI sites as promoter <i>Plsd90</i> respectively. The resulting vectors were designated as pJH6c (A). The strategy of <i>Fluc</i> cloning is described in the methods section.</p