The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LI...

background brucellosis is an important cosmopolitan infection disease caused by organisms belonging to the genus brucella. the cgt gene (cyclic β-1, 2 glucan transporter gene) is a virulent factor in brucella genus. the present study was conducted with the aim of cloning and expression of brucella cgt gene. materials and methods brucella melitensis cgt gene was amplified from extracted chromoso...

Production of recombinant proteins is the starting point for biochemical and biophysical analyses and requires methodology to efficiently proceed from gene sequence to purified protein. While optimized strategies for the efficient cloning of single-gene fragments for bacterial expression is available, efficient multiple DNA fragment cloning still presents a challenge. To facilitate this step, w...

Hyperthermophile Pyrococcus furiosus grows optimally near 100°C and is an important resource of many industrial and molecular biological enzymes. To study the structure and function of P. furiosus proteins at whole genome level, we constructed expression plasmids of each P. furiosus gene using a ligase-independent cloning method, which was based on amplifying target gene and vector by PCR using...

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The express...

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five 'pQLink' vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expr...

Aeromonas hydrophila is a gram-negative bacterium which associated with gastrointestinal diseases and septicaemia. This pathogenic bacterium has several virulence factors ranging from pili to the excreted protein which called (Aerolysin) with minor and major effects, respectively. Additionally, Aeromonas hydrophila is a widely distributed bacterium that commonly causes ulcers in cyprinid fish s...

objective: in this project, our aim was to construct a novel expressing vector harboring a new sequence from overlapping region of ns3 gene of hcv from infected iranian patient. materials and methods: the partial ns3 (pns3) gene was amplified by nested-rt-pcr method using sera of hcv infected patients harboring genotype 1a. after purification and cloning the pns3 into ta-cloning vector, the be...

the aim of this study was to clone the serine alkaline protease-encoding gene from bacillus subtilis 168. this protease, which can have many applications especially in detergent, may be industrially an important enzyme. for the amplification of the gene, pcr was performed with a pair of primers specifically designed for this purpose. electrophoresis of the pcr product showed the expected band o...

Background: Thrombopoietin (TPO) is an important cytokine that has a critical role in regulating hematopoietic stem cells (HSCs) proliferation and megakaryocyte differentiation. Because of scares amount of this protein in human plasma, in many biotechnological centers around the world, recombinant production of this protein has been carried out. This study was aiming to gene cloning and express...