Rapid cloning by homologous recombination<i>in vivo</i>

Bubeck, Peter; Winkler, M; Bautsch, Wilfried

doi:10.1093/nar/21.15.3601

Cited by 135 publications

(134 citation statements)

References 2 publications

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“…For this Pfu polymerase, the primers 5Ј-GAG-AAGACCGGTCTTGC and 5Ј-TTTGATCTTCTCAAGTT-GTCG were used. The PCR products were co-transformed into CaCl 2 -competent E. coli TOP10 cells, utilizing the endogenous recombinase activity of E. coli to recombine the fragments (13). The protein coding part of the resulting expression vector, pPICZ-hisMPGES, was sequenced for verification.…”

Section: Methodsmentioning

confidence: 99%

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

Hammarberg

Hámberg

Wetterholm

et al. 2009

Journal of Biological Chemistry

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Microsomal prostaglandin E synthase type 1 (mPGES-1) converts prostaglandin endoperoxides, generated from arachidonic acid by cyclooxygenases, into prostaglandin E 2 . This enzyme belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral membrane proteins, and because of its link to inflammatory conditions and preferential coupling to cyclooxygenase 2, it has received considerable attention as a drug target. Based on the high resolution crystal structure of human leukotriene C 4 synthase, a model of mPGES-1 has been constructed in which the tripeptide co-substrate glutathione is bound in a horseshoe-shaped conformation with its thiol group positioned in close proximity to Arg-126. Mutation of Arg-126 into an Ala or Gln strongly reduces the enzyme's prostaglandin E synthase activity (85-95%), whereas mutation of a neighboring Arg-122 does not have any significant effect. Interestingly, R126A and R126Q mPGES-1 exhibit a novel, glutathione-dependent, reductase activity, which allows conversion of prostaglandin H 2 into prostaglandin F 2␣ . Our data show that Arg-126 is a catalytic residue in mPGES-1 and suggest that MAPEG enzymes share significant structural components of their active sites. Prostaglandin E 2 (PGE 2 )3 is an abundant lipid mediator that signals via four receptors (EP1 to -4), resulting in an array of important biological actions in physiology as well as pathophysiology (1, 2). Biosynthesis of PGE 2 proceeds from arachidonic acid, which is converted to the unstable endoperoxide, prostaglandin H 2 , by cyclooxygenase type 1 and 2. PGH 2 is further isomerized into PGE 2 by three distinct enzymes, cytosolic PGE synthase, microsomal PGE synthase type 1 (mPGES-1), and microsomal PGE synthase type 2 (3-5). Although cytosolic PGE synthase and microsomal PGE synthase type 2 seem to provide a basal synthesis of PGE 2 , mPGES-1 appears to account for PGE 2 synthesis under proinflammatory conditions. Thus, mPGES-1 is up-regulated by mitogens and cytokines and is functionally coupled to cyclooxygenase type 2 (4, 6). Due to its key role in PGE 2 synthesis, mPGES-1 has attracted attention as a potential drug target in the areas of inflammation, pain, fever, and cancer (7).mPGES-1 is a member of the MAPEG superfamily of enzymes (8), which also includes two key proteins in the leukotriene cascade, viz. 5-lipoxygenase-activating protein and leukotriene C 4 synthase (LTC4S). Since all MAPEG members are integral membrane proteins, structural information on this family has been scarce. Recently, however, significant progress has been made in this area, and several high and low resolution structures have been solved by three-dimensional as well as two-dimensional crystallography (9 -12). The crystal structures of human LTC4S clearly provided the most detailed structural information, inter alia a unique, horse-shoe shaped binding conformation of GSH, a hydrophobic crevice presumably binding the lipid substrate leukotriene A 4 , and an Arg residue, possibly involved in...

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Section: Methodsmentioning

confidence: 99%

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

Hammarberg

Hámberg

Wetterholm

et al. 2009

Journal of Biological Chemistry

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“…The sequence of the sense and antisense primers was as follows: 5Ј-GGCCTGGTGCCGCGCGGCAGCGCGGAG-TACGACTTGACTAC-3Ј and 5Ј-CTCAGCTTCCTTTCGGGCTTCTTCA-GTAGAAGCCAGAATC-3Ј. The PCR product was cloned by homologous recombination (27) in pET-15b vector (Novagen) digested with NdeI and BamHI. For the HSPC021 bacterial expression vector, the cDNA was amplified by PCR as for pSGF-HSPC021 and inserted into the BglII site of the bacterial expression vector pFLAG-2 (Sigma).…”

Section: Methodsmentioning

confidence: 99%

The Human Protein HSPC021 Interacts with Int-6 and Is Associated with Eukaryotic Translation Initiation Factor 3

Morris¹,

Réty²,

Ferro³

et al. 2001

Journal of Biological Chemistry

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The Int-6 protein has been shown to be a subunit of eukaryotic translation initiation factor 3 (eIF3) and to play a role in the control of cell growth. By immunoprecipitation experiments and mass spectrometry analyses, we identified a human protein previously known as HSPC021 that is associated with Int-6. Exposure of Jurkat cells to the phosphatase inhibitor H 2 O 2 triggers a marked phosphorylation on tyrosine of HSPC021. Several experiments were performed to evaluate whether this protein is associated with eIF3. It was observed that HSPC021 coelutes with Int-6 and eIF3 in gel filtration, coimmunoprecipitates with eIF3, and is incorporated into eIF3 both in rabbit reticulocyte lysates and in COS7 cells. A direct protein-protein interaction occurs between HSPC021 and Int-6, but the analysis of different mutants of HSPC021 indicated that a larger region of the protein is necessary for incorporation into eIF3 as compared with binding to Int-6. Taken together, our results establish that HSPC021 is tightly associated with the mammalian translation initiation factor eIF3. Analysis of the primary sequence of HSPC021 from different species revealed the presence of a tetratricopeptide repeat, a proteasome-COP9 (constitutive photomorphogenesis 9) signalosome-initiation factor 3 domain along with a Pumilio FBF repeat. These protein motifs are also present in subunits of eIF3, of the lid of the 26 S proteasome, and of the COP9 signalosome.

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“…or pGEX-VP1-S4 using in vivo homologous recombination (Bubeck et al 1993, Jones 1994, Oliner et al 1993, Parrish et al 2004. Briefly, purified AfeI-linearised pGEX-VP1-S1S4 or pGEX-VP1-S4 was mixed with purified DNA fragment at an optimised ratio of 1 to using the heat shock method (Sambrook and Russell 2001).…”

Section: Generation Of Modular Constructsmentioning

confidence: 99%

“…The purified DNA fragment was cloned into AfeI-linearised vector pGEX-VP1-S1S4 using the in vivo homologous recombination (Bubeck et al 1993, Jones 1994, Oliner et al 1993, Parrish et al 2004, as described in Section 4.2.1.…”

Section: Construct Vp1-h3-h190-h190-4ementioning

confidence: 99%

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

Anggraeni¹

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A promising alternative to replace the current egg-or cell culture-based technology for vaccine production from live viruses is virus-like particle (VLP) technology based on a microbial platform. VLPs are macromolecular assemblies of viral capsid proteins, which have been shown to tolerate insertion of antigen modules via genetic recombinant technology, yielding modular VLPs. Many studies on modular VLPs presume that when a peptide antigen element is taken out from the intact proteins and then modularised on VLPs, it is unable to fold into its native structure. However, until now, presentation of a peptide antigen element on a VLP and the impact of the display strategy to present the antigen element on the quality of the resulting antibodies (i.e. the ability of the antibodies to recognise the intact protein) are not fully understood. This thesis aims to understand the underlying fundamentals regarding modularisation of peptide antigen elements on VLPs for induction of high-quality antibodies. A hypervariable receptor-binding domain, Helix 190 (H190), from the hemagglutinin protein of influenza A virus was used as a model for modularisation on VLPs from murine polyomavirus (MuPyV) VP1 protein. Four major findings are presented. Firstly, two display strategies, i.e. arraying of H190 in tandem repeats and the use of helix promoter elements, were shown to display H190 in its immunogenic form equally. However, modularisation using tandem repeat display induced antibodies of a higher quality than modularisation using helix promoter elements.Secondly, the quality of antibodies induced by the tandem repeat display bearing two copies of H190 was optimum, thus no significant improvement was observed following the use of adjuvant or increasing the copy number of H190. Additionally, the increase in the copy number of H190 was shown to reduce the assembly capability and solubility of modular VP1 in an environment that was optimised for wild-type VP1. Thirdly, this thesis shows the novel finding in the use of flanking ionic elements to stabilise VLP precursors, termed as capsomeres, bearing two copies of H190 containing a hydrophobic stretch, which caused aggregation. Fourthly, the first steps towards obtaining the atomic crystal structure of presented H190 on a modular protein were performed, i.e. a mild and satisfactory laboratory process was developed to achieve high-purity modular VP1 capsomeres, unattainable using previously established expression and purification process of wild-type MuPyV VP1. This thesis shows a step forward towards understanding the presentation of a peptide antigen element on a VLP that enables induction of highquality antibodies, and towards VLP engineering to manipulate the aggregation and solubility of modular VP1. VLP technology based on a microbial platform presented here is iii a potentially safe and effective alternative vaccine candidate that targets a hypervariable peptide antigen element. The speed of the microbial platform allows a rapid response to the hypervariability of the pe...

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Rapid cloning by homologous recombinationin vivo

Cited by 135 publications

References 2 publications

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

Mutation of a Critical Arginine in Microsomal Prostaglandin E Synthase-1 Shifts the Isomerase Activity to a Reductase Activity That Converts Prostaglandin H2 into Prostaglandin F2α*

The Human Protein HSPC021 Interacts with Int-6 and Is Associated with Eukaryotic Translation Initiation Factor 3

Engineering of virus-like particles for alternative vaccine candidate targeting a hypervariable peptide antigen element

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