Integration of PCR Fragments at Any Specific Site within Cloning Vectors without the Use of Restriction Enzymes and DNA Ligase

Geiser, Martin; Cèbe, Régis; Drewello, Delia; Schmitz, Rita

doi:10.2144/01311st05

Cited by 228 publications

(200 citation statements)

References 3 publications

(3 reference statements)

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“…Similar constructs lacking domain A1 (Glu-1260-Gly-1479) and͞or domain A3 (Glu-1673-Gly-1874), or with the mutation R1597Q in domain A2, were made by a PCR-based method using the restriction endonuclease DpnI (Stratagene) (21) and primer 5 (5Ј-gctgatctccgaggaggacctgccggggctcttgggggtttcgac-3Ј), primer 6 (5Ј-gctgcagaggtgctgctccggagtggcggccgctcaccatcacc-3Ј), or primer 7 (5Ј-gtcagccagggtgaccaggagcaggcgcccaac-3Ј), respectively. The vWF signal peptide (Met-1-Cys-22) was amplified from pSVHvWF1 with primer 8 (5Ј-ctttaagaaggagcccttcaccatgattcctgccagatttgccg-3Ј) and primer 9 (5Ј-cctcggagatcagcttctgctcacaaagggtccctggcaaaatg-3Ј) and inserted into each clone by PCR (22). The DNA sequence of clones was confirmed, and This paper was submitted directly (Track II) to the PNAS office.…”

Section: Methodsmentioning

confidence: 98%

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

Nishio

Anderson

Zheng

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

142

135

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von Willebrand factor (vWF) is a multimeric plasma glycoprotein with three tandem A domains. Domains A1 and A3 bind to platelet glycoprotein Ib␣ (GPIb␣) and collagen, respectively. Domain A2 contains the Tyr-1605-Met-1606 bond that is cleaved by the metalloprotease ADAMTS13, and this reaction inhibits platelet thrombus growth. Fluid shear stress increases the rate of cleavage, suggesting that productive interaction with ADAMTS13 requires conformational changes within or near domain A2. The influence of the adjacent A1 and A3 domains was assessed by mutagenesis of a recombinant substrate consisting of domains A1A2A3. Deletion of domain A3 did not affect cleavage by ADAMTS13, whereas deletion of domain A1 increased the rate of cleavage Ϸ10-fold. Similar effects were observed with plasma ADAMTS13 and recombinant ADAMTS13 truncated after the spacer domain. Digestion of A1A2A3 by plasma ADAMTS13 was enhanced to a similar extent by a recombinant mutant fragment of platelet GPIb␣ that binds with high affinity to domain A1 or by heparin. Heparin also increased the digestion of purified plasma vWF. Neither GPIb␣ nor heparin increased the cleavage of substrate A2A3 that lacks domain A1. The results suggest that vWF domain A1 inhibits the cleavage of domain A2, and that inhibition can be relieved by interaction of domain A1 with platelet GPIb␣ or certain glycosaminoglycans. Thus, binding of vWF to its major physiological ligands may promote the feedback inhibition of platelet adhesion by stimulating the cleavage of domain A2 by ADAMTS13 independent of fluid shear stress.

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Section: Methodsmentioning

confidence: 98%

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

Nishio

Anderson

Zheng

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

142

135

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“…The gene was digested out of the synthetic vector using designed PstI and HindIII restriction sites and cloned into the pMal-c2x expression vector encoding a maltose-binding protein fusion product. Into this construct, Fn3 clones were inserted by amplification of Fn3 genes out of their own expression vectors and using the purified amplification products as primers for a QuikChange TM insertion similar to the protocol described by Geiser et al (37). The linker between Fn3 and rGel was modified to consist strictly of the amino acids encoded by the necessary restriction sites for binder cloning and a G 4 S sequence.…”

Section: Methodsmentioning

confidence: 99%

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Pirie¹,

Hackel²,

Rosenblum

et al. 2011

Journal of Biological Chemistry

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Gelonin-based immunotoxins vary widely in their cytotoxic. Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ϳ5 ؋ 10 6 toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.Immunotoxins are a promising approach to the targeted delivery of highly potent, cancer-specific, cytotoxic agents. Immunotoxins are frequently composed of a targeting moiety (derived from antibodies or other cell-binding proteins) either chemically conjugated or genetically fused to highly cytotoxic plant or bacterial protein toxins. Clinical success for immunotoxins has been mostly limited to hematological malignancies due to transport limitations in solid tumors (1). Such limitations have been extensively studied experimentally (2) and with several computational models (3, 4).The potency of a particular immunotoxin is dependent on the ability to deliver the toxin to the cytoplasm, which is commonly considered to be the rate-limiting step. For some native toxins such as ricin, intracellular delivery is achieved through lectin binding, followed by internalization and toxin release with membrane fusion or retrograde trafficking (5). Immunotoxins attempt to recreate this scenario by replacing the indiscriminate lectin binding with cancer-specific antigen binding as a means of targeting and internalization (6). Subsequent intracellular trafficking, release, and endosomal escape are often achieved using existing toxin characteristics, translocation domains, protease cleavage sites, disulfide bonds, and/or signaling peptides (7-10). However, the inclusion of toxins with domains facilitating cytoplasmic access can also lead to increased nonspecific toxicity in vivo (11,12).Gelonin is a plant toxin and classified as a type I ribosomeinactivating protein because it lacks any cell-binding or cytoplasmic delivery domains. Recombinant gelonin (rGel) 2 is an ϳ30-kDa N-glycosidase with activity similar to the ricin A chain but exhibiting better stability and lower immunogenicity (13,14). The use of rGel in tumor-targeted cytotoxic agents has been well studied (15, 16). Furthermore, rGel has been shown to be active without cleavage from the binding domain and without negative impact on the targeting agent's pharmacokinetics (...

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“…At the 5Ј-end of each oligonucleotide, the nucleotides necessary for the integration of the final PCR fragment into the template baculovirus transfer vector were added by following the rules we described (24), and the PCR fragment was finally integrated into the BacPAK8 TM transfer vector (Clontech) resulting in plasmid pXI392. The correctness of the construction was verified by DNA sequence analysis.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Ligand-independent Transcriptional Activation of the Human Estrogen-related Receptor α (ERRα)

Kallen¹,

Schlaeppi

Bitsch³

et al. 2004

Journal of Biological Chemistry

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195

109

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The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor ␣ (ERR␣, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1␣ (PGC-1␣) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERR␣ LBD, solved to a resolution of 2.5 Å, and the first structure of a PGC-1␣ complex. The putative ligand binding pocket (LBP) of ERR␣ is almost completely occupied by side chains, in particular with the bulky side chain of Phe 328 (corresponding to Ala 272 in ERR␥ and Ala 350 in estrogen receptor ␣). Therefore, a ligand of a size equivalent to more than ϳ4 carbon atoms could only bind in the LBP, if ERR␣ would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERR␣. The interactions of PGC-1␣ with ERR␣ also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1␣ peptide containing this nuclear box motif from the L3 site binds ERR␣ LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1␣ are present.Nuclear hormone receptors (NRs) 1 are transcription factors that control essential developmental and physiological pathways (1). Although the transcriptional activity of NRs is often regulated by specific ligands, several members of the superfamily have no known natural ligands and are therefore referred to as orphan NRs (2). Estrogen-related receptor ␣ (ERR␣; NR3B1) was the first orphan NR to be identified on the basis of its similarity with estrogen receptor ␣ (ER␣; NR3A1) (3). ERR␣ and its relatives ERR␤ (NR3B2) and ERR␥ (NR3B3) form a small family of orphan NRs that are evolutionarily related to the estrogen receptors ER␣ and ER␤. ERRs preferentially bind to DNA sites composed of a single half-site preceded by three nucleotides with the consensus sequence TNAAGGTCA, referred to as an ERR response element. It has been shown that ERR␣ also efficiently binds to estrogen response elements and that these receptors share common target genes (4). This observation was further supported by studies demonstrating cross-talk between the ER and ERR pathways (reviewed in Ref. 5). The most striking feature observed in the phenotype of mice lacking ERR␣ is their resistance to high fat diet-induced obesity and the impaired activity of enzymes implicated in lipid metabolism. This finding led to the hypothesis that ERR␣ could be implicated in obesity or metabolic diseases (6). A function of ERR␣ on bone metabolism has also been suggested (7,8). Finally, recent publications show that ERR␣ and ERR␥ are associated with biomarkers of breast cancer and further emphasize the importance of ER-ERR cross-talk (9...

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Integration of PCR Fragments at Any Specific Site within Cloning Vectors without the Use of Restriction Enzymes and DNA Ligase

Cited by 228 publications

References 3 publications

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

Binding of platelet glycoprotein Ibα to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13

Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties

Evidence for Ligand-independent Transcriptional Activation of the Human Estrogen-related Receptor α (ERRα)

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