Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycl...

We have produced a murine monoclonal antibody (F43 A1D2) that binds to the cell surface of both rat islet tumor cells (RINm clone 5F and RINm clone 14B) and normal rat islet cells. This antibody is cytotoxic in the presence of complement for RIN tumor cells as well as A, B, and D pancreatic polypeptide rat islet cells. Antibody A1D2 does not bind to rat thymus cells, pancreatic acinar cells, or...

A comprehensive gene collection for S. oneidensis was constructed using the lambda recombinase (Gateway) cloning system. A total of 3584 individual ORFs (85%) have been successfully cloned into the entry plasmids. To validate the use of the clone set, three sets of ORFs were examined within three different destination vectors constructed in this study. Success rates for heterologous protein exp...

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the s...

The work was to explore the feasibility of protein affinity purification using ligand isolated from phage library. Reteplase was used as the model protein and a humanized semi-synthetic single chain fragment variable phage library as the source of ligand. After four rounds of biopanning, reteplase-specific phage clones were greatly enriched. The scFv gene from the best phage clone was inserted ...

The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of station...

Abstract DNA isolation is a fundamental technique for all molecular biology laboratories. Depending on the plant species, can be challenging. In particular, adapted protocols rarely exist tree species which are not used as standard model organisms. Here, we describe flexible protocol that works 59 in modular system. It based an ATMAB-containing extraction buffer to proteinase K and/or boric aci...

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene down...

Functional screening of a metagenomic library of marine sediment revealed an amylolytic clone BTM109. This report states the purification and characterization of a moderately halotolerant α-amylase, with more than 51% activity in 2.5 M NaCl. The molecular mass of purified protein was determined to be 55.7 kDa by MALDI-TOF MS. The optimum pH for enzyme activity was pH 7 and temperature for maxim...

Opioid receptors were one of the first class of cell surface receptors to be identified using in vitro binding assays with brain tissue, but for many years efforts to purify and clone them were unsuccessful because of their sensitivity to detergents, their heterogeneity, and the lack of a simple biochemical assay for their function (Loh and Smith 1990). Several laboratories, including the autho...