A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site

Stols, Lucy; Gu, M.; Dieckman, Lynda; Raffen, Rosemarie; Collart, F.; Donnelly, Mark I.

doi:10.1006/prep.2001.1603

Cited by 499 publications

(460 citation statements)

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“…A His 6 -Nipped-B protein fusion containing Nipped-B residues 1 to 409 (GenBank AF114160) was expressed in E. coli using the pMCSG-7 vector (Stols et al 2002) and purified under denaturing conditions. Insoluble purified protein was washed and suspended in phosphatebuffered saline (PBS) and used to immunize a guinea pig and a rabbit (Pocono Rabbit Farm and Laboratory, Canadensis, PA).…”

Section: Nipped-b Pds5 and Rad21 Antibodiesmentioning

confidence: 99%

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

et al. 2007

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Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B's functions, we compared the effects of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. We also examined association of Nipped-B and cohesin with somatic and meiotic chromosomes by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes

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Section: Nipped-b Pds5 and Rad21 Antibodiesmentioning

confidence: 99%

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

et al. 2007

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“…1, Table 1) is derived from the simple his 6 -tag-TEV-site vector, pMCSG7 [13], which has been used routinely for the production of proteins within the MCSG.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

“…The vector pMCSG9 was constructed by inserting the gene encoding MBP into the KpnI site of vector pMCSG7 [13]. The MBP encoding region was generated by PCR using plasmid pRK793 [17] as template (a generous gift from David Waugh) and the primers 5′-TTTTAGATCTGATGTCCCCTATACTAGGTTATTGG and 5′-TTTTGGTACCTGGGATATCGTAATCATCCGATTTTGGAGGATGGT (purchased from the Howard Hughes Medical Institute-Keck Laboratory of Yale University, New Haven, CT).…”

Section: Construction Of Pmcsg9 and Pmcsg19mentioning

confidence: 99%

“…The material was then treated with 40 U LIC-qualified T4 DNA polymerase (Novagen, Madison, WI) and 2.5 mM dGTP in a volume of 40 μl in 1× commercial buffer supplemented with 5 mM DTT. Genes were amplified by PCR with primers encoding the LIC overhang [13] (sense: TACTTCCAATCCAATGCX followed by the genes' N-terminal sequences; antisense: TTATCCACTTCCAATG followed by the complement of a stop codon and of the C-terminus of the gene), purified with a QIAQuick PCR purification kit (Qiagen), and treated with T4 polymerase as described above except in the presence of dCTP. Following annealing of 30-50 ng of this material with 15 ng LIC-prepared vector, the resulting plasmids were transformed into DH5α, and plasmids prepared from these transformants were introduced into BL21(DE3) containing the plasmid pRK1037 [15] (Science Reagents, Inc.).…”

Section: Ligation-independent Cloning Into Pmcsg Vectorsmentioning

confidence: 99%

“…1, Table 1) is derived from the simple his 6 -tag-TEV-site vector, pMCSG7 [13], which has been used routinely for the production of proteins within the MCSG. The new vector applies strategies developed by Waugh and colleagues [14,15] to the problems outlined above.…”

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confidence: 99%

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An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Donnelly

Zhou

Millard

et al. 2006

Protein Expression and Purification

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161

172

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Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his 6 -tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his 6 -tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his 6 -tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his 6 -site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his 6 -tagged target protein.Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his 6 -tag. KeywordsHigh-throughput; Structural genomics; Maltose-binding protein; TVMV protease; Ligationindependent cloningThe burgeoning genomic information now available makes vast numbers of proteins accessible for structural and functional studies, and many large-scale projects have developed automated protocols for amplifying, cloning, and expressing genes, and for screening proteins for desirable properties [1][2][3][4][5]. Similar strides have been made in streamlining protein purification, but production of sufficient material for detailed structural and functional characterization remains labor-intensive and time-consuming [3,4,6,7]. Typically, purification is facilitated by fusing proteins to affinity tags, most commonly a his-tag, which allows purification by immobilized metal-ion affinity chromatography (IMAC,[8]). Additional tags are often attached to improve proteins' solubility, such as maltose-binding protein (MBP) [2][3][4]9,10]. In typical protein production pipelines, the resulting fusion proteins are first screened for ☆ This manuscript has been created by the University of Chicago as operator of Argonne National Laboratory under Contract No.W-31-109-ENG-38 with the US Department of Energy. The US government retains for itself, and others acting on its behalf, a paid-up, nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the government. The US Government's right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript solubility, then purified by semi-robotic p...

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Structural genomics – expanding protein structural universe

Joachimiak

2005

Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics

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Structural Genomics has emerged as a multidisciplinary and international effort to expand the universe of protein folds by rapidly determining novel structures. This initiative takes advantage of the genome sequencing projects and technological advances in genomic analysis, molecular biology, proteomics, and structural biology. The international Structural Genomics efforts were initiated, in part, to achieve a more rapid understanding of sequence/structure relationships with the ultimate goal to make it feasible to predict an accurate 3D protein structure on the basis of sequence alone, and to elucidate novel biological function directly from primary genomic sequence. A number of Structural Genomics centers have been established to test and improve existing and develop new technologies. These centers developed the protein structure determination pipelines that include target selection, gene cloning and expression, protein and crystal production, structure determination, and structural and functional analysis. These pipelines are capable of determining hundreds of novel protein structures. Structural Genomics is rapidly expanding the universe of protein folds, creating new reagents, comprehensive libraries of expression clones, and databases for broad use. New high‐throughput technologies supported by robotics and automation in molecular biology, protein expression, purification, crystallization, and structure determination are being disseminated to the public and, therefore, contribute to the acceleration of new discoveries in biology. Ultimately, Structural Genomics will become a major component of and contributor to the next structural biology challenge – visualization of all major subcellular macromolecular assemblies of the cell.

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A New Vector for High-Throughput, Ligation-Independent Cloning Encoding a Tobacco Etch Virus Protease Cleavage Site

Cited by 499 publications

References 14 publications

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

Structural genomics – expanding protein structural universe

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