Vectors for selective expression of cloned DNAs by T7 RNA polymerase

Rosenberg, Alan H.; Lade, Barbara N.; Dao-shan, Chui; Lin, Shi‐Ming; Dunn, John J.; Studier, F. William

doi:10.1016/0378-1119(87)90165-x

Cited by 1,462 publications

(897 citation statements)

References 16 publications

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“…This plasmid pS67P was cut with BamHI, and the 0.4-kb fragment was inserted into the BamHI site of the expression vector pAR3039. The resulting plasmid pARDS18-1 produced a large quantity of polypeptide in E. coli BL21 (DE3) pLysS (Rosenberg et al, 1987). The M.W.…”

Section: Preparation Of Bacterial-made Dskl Proteins and Antiseramentioning

confidence: 99%

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

Takeuchi

Yanagida

1993

MBoC

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The fission yeast dskl+ gene, a multicopy suppressor for cold-sensitive disi mutants, encodes a novel 61-kd protein kinase. It is a phosphoprotein, and phosphoserine is the major phosphorylated amino acid. Hyperphosphorylation of dskl causes a mobility shift, resulting in two dskl-specific protein bands. The phosphorylation pattern is strikingly altered when cell cycle progression is delayed or arrested. The slowly migrating phosphorylated form is prominent in mitotically arrested cells, and the fast migrating form is enriched in interphasearrested cells. dskl is a protein kinase. It auto-phosphorylates as well as phosphorylates myelin basic protein (MBP). Phosphotyrosine as well as phosphoserine/threonine were found in autophosphorylation, but no tyrosine phosphorylation occurs when MBP was used as the substrate. The dskl immunoprecipitates from mitotically arrested cells have a several-fold higher kinase activity than that from wild type. The haploid gene disruptant is viable, indicating that the dskl+ gene is non-essential for viability. High dosage of dskl+, however, strongly delays the G2/M progression. Immunofluorescence microscopy using anti-dskl antibody shows that localization pattern of dskl protein strikingly alters depending on cell cycle stages. In G2-arrested cells, dskl locates in the cytoplasm, whereas in mitotically arrested cells, nuclear stain is intense. In wild-type cells, nuclear stain is seen only in mitotic cells. Hence dskl protein may play an important role in mitotic control by altering cellular location, degree of phosphorylation and kinase activity. We discuss possible roles of dskl kinase as an add-on regulator in mitosis.

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Section: Preparation Of Bacterial-made Dskl Proteins and Antiseramentioning

confidence: 99%

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

Takeuchi

Yanagida

1993

MBoC

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“…), Ea,,:h fral/mem harbours the introduced mutations either at tl~e 3" or 5' site, They were isolated from a 6% polyacrylamide gel [25i. Tire complete SPL fragment encoding tire desired mutations and a restored Bglll site wax generated by performing a second PCR using primer 393, 1956 on SPI fragments A and B, The PCR prodttct was extracted with pbenolA;hloroform, digested with BamHI and Bglll, gel purified and cloned into pAR 3039 [28] to yield pAR-SPI-Kox 29. Standard PCPt conditions were applied [251, Introduced mutattons were verified by dideoxy sequence an:tlysis,…”

Section: Oligonucleotidesmentioning

confidence: 99%

Determination of DNA binding specificities of mutated zinc finger domains

Thiesen¹,

Bach²

1991

FEBS Letters

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By substituting non conserved amino acids present in the postulated α‐helical region of zinc finger domains, we demonstrated that Cys2/His2 type zinc finger domains could be targeted to new DNA binding sites. The putative α‐helical region of the second SPI zinc finger (SLH) was replaced by amino acids (SLH) occurring in analogous zinc finger positions of human zinc finger protein Kox 29. The DNA binding specificity of the FPLC purified chimaeric protein (SPI‐Kox 29) was determined by use of the target detection assay (TDA). Chimaeric protein SPI‐Kox 29 wax subjected to randomized oligonucleotides (GGG NNNN GGC) that were designed on the basis that each SPI zinc finger interacts with 3–4 nucleotides concerning its cognate target site GGG GCGG GGC. By this analysis the DNA binding specificity of SPI‐Kox 29 was shown to have switched from the cognate SPI binding site to GGG GGTG GGC. Structure‐function analysis of this type should facilitate the determination of DNA binding specificities for any individual zinc finger of interest.

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“…Development of affinity tags such as the maltose-binding tag (di Guan et al, 1988;Maina et al, 1988), the polyhistidine tags of the pET vector series, (Rosenberg et al, 1987;Studier & Moffatt, 1986;Studier et al, 1990), and the IMPACT® intein chitinCorrespondence should be sent to Jorge C. Escalante-Semerena, Department of Bacteriology, University of Wisconsin, 1550 Linden Dr, Madison, WI 53706 USA; E-mail: escalante@bact.wisc.edu, phone: (608) 262-7379, fax: (608) 265-7909 or to Ivan Rayment, Department of Biochemistry, University of Wisconsin, 433 Babcock Dr, Madison, WI, 53706; E-mail: Ivan_Rayment@biochem.wisc.edu, phone: (608) 262-0437.. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.…”

Section: Introductionmentioning

confidence: 99%

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

Rocco

Dennison

Klenchin

et al. 2008

Plasmid

120

121

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We describe the construction and use of two sets of vectors for the over-expression and purification of protein from E. coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His 6 ) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His 6 and maltose-binding protein tags in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

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Vectors for selective expression of cloned DNAs by T7 RNA polymerase

Cited by 1,462 publications

References 16 publications

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

Determination of DNA binding specificities of mutated zinc finger domains

Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli

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