Expression and Purification of Recombinant Human Granzyme B from Pichia pastoris

Sun, Jiuru; Bird, Catherina H.; Buzza, Marguerite S.; McKee, Katherine; Whisstock, James C.; Bird, Phillip I.

doi:10.1006/bbrc.1999.0989

Cited by 58 publications

(45 citation statements)

References 25 publications

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“…The following were used for confocal microscopy: mouse monoclonal anti-cytochrome c Recombinant human GzmB, hGzmA and mouse GzmA were produced as previously described. 36 Recombinant mouse GzmA (mGzmA) WT and active-site mutant were produced by amplifying the mGzmA coding sequence from a plasmid template by PCR using primers that replaced the signal sequence with a combined FLAG tag and enterokinase cleavage site at the N-terminus and added hexahistidine tag at the C-terminus. The product was the cloned into pPIC9, transformed into Pichia.…”

Section: Methodsmentioning

confidence: 99%

“…pastoris and analysed as previously described. 36 For protein production, P. pastoris expressing mGzmA were grown at 30 1C for 36 h, and then allowed to settle for 12 h at room temperature; growth medium was replaced with induction medium containing 3% methanol and 0.5 M arginine. Cells were induced at 23 1C for 60 h and again allowed to settle for 12 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were induced at 23 1C for 60 h and again allowed to settle for 12 h at room temperature. mGzmA was then purified from induction medium as previously described, 36 except at pH 8.0. Once activated, mGzmA was separated from the enterokinase with a further SP-sepharose column, the final activated mGzmA was concentrated and stored at À 70 1C.…”

Section: Methodsmentioning

confidence: 99%

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Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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Human and mouse granzyme (Gzm)B both induce target cell apoptosis in concert with pore-forming perforin (Pfp); however the mechanisms by which other Gzms induce non-apoptotic death remain controversial and poorly characterised. We used timelapse microscopy to document, quantitatively and in real time, the death of target cells exposed to primary natural killer (NK) cells from mice deficient in key Gzms. We found that in the vast majority of cases, NK cells from wild-type mice induced classic apoptosis. However, NK cells from syngeneic Gzm B-deficient mice induced a novel form of cell death characterised by slower kinetics and a pronounced, writhing, 'worm-like' morphology. Dying cells initially contracted but did not undergo membrane blebbing, and annexin-V staining was delayed until the onset of secondary necrosis. As it is different from any cell death process previously reported, we tentatively termed this cell death 'athetosis'. Two independent lines of evidence showed this alternate form of death was due to Gzm A: first, cell death was revealed in the absence of Gzm B, but was completely lost when the NK cells were deficient in both Gzm A and B; second, the athetotic morphology was precisely reproduced when recombinant mouse Gzm A was delivered by an otherwise innocuous dose of recombinant Pfp. Gzm A-mediated athetosis did not require caspase activation, early mitochondrial disruption or generation of reactive oxygen species, but did require an intact actin cytoskeleton and was abolished by latrunculin B and mycalolide B. This work defines an authentic role for mouse Gzm A in granule-induced cell death by cytotoxic lymphocytes.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

et al. 2013

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“…Recombinant wild type and RAH GrB were produced as zymogens in Pichia pastoris and activated following methods described previously (16), except that the His tag was incorporated upstream of the enterokinase cleavage site, which allowed simultaneous activation of the zymogen and removal of the His tag. The RAH mutations were introduced into the wild type GrB sequence using an overlapping PCR strategy.…”

Section: Methodsmentioning

confidence: 99%

“…In vitro transcription and translation of mouse procaspase 3 and Bid was carried out as described (16), using the Promega TNT system.…”

Section: Methodsmentioning

confidence: 99%

Granzyme B Encoded by the Commonly Occurring Human RAH Allele Retains Pro-apoptotic Activity

Sun

Bird

Thia

et al. 2004

Journal of Biological Chemistry

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A key function of human granzyme B (GrB) is to induce apoptosis of target cells in conjunction with perforin. The RAH allele is the first documented variant of the human GrB gene, occurs at a frequency of 25-30%, and encodes three amino acid substitutions (Q48R, P88A, and Y245H). It was initially reported that RAH GrB is incapable of inducing apoptosis, but here we show that it has essentially identical proteolytic and cytotoxic properties to wild type GrB. Recombinant RAH and wild type GrB cleave peptide substrates with similar kinetics, are both capable of cleaving Bid and procaspase 3, and are equally inhibited by proteinase inhibitor 9, an endogenous regulator of GrB. Furthermore, cytotoxic lymphocytes from RAH heterozygotes and homozygotes have no defect in target cell killing, and in vitro RAH GrB and wild type GrB kill cells equally well in the presence of perforin. We conclude that the RAH allele represents a neutral polymorphism in the GrB gene.Cytotoxic lymphocytes (CLs) 1 play a central role in cellmediated immunity by destroying virus-infected, foreign, or tumor cells (1). They also contribute to the pathology of autoimmune diseases such as diabetes and to graft versus host disease following bone marrow transplantation (1). On contact with a target cell, the CL releases cytotoxins such as perforin, proteases (granzymes), and death ligands from secretory granules into the intercellular space. Target cell death then follows one of two pathways: activation of death receptors on the target cell surface coupled to caspase activation within the cell or uptake of granzymes in a perforin-dependent manner that leads to caspase activation and intracellular proteolysis (2). In the latter pathway, perforin and granzymes are endocytosed by the target cell, and perforin mediates release of granzymes into the cytoplasm (3). Caspase activation, loss of mitochondrial membrane potential, proteolysis of key house-keeping proteins, DNA degradation, and the disintegration of cellular structures follow rapidly.Human CLs produce granzyme A (GrA) and granzyme B (GrB), two cytotoxic granule serine proteases that activate different death pathways (2). GrA cleaves after basic residues, appears to cause single-stranded DNA breaks, and disrupts repair mechanisms in the target cell, leading to caspase-independent death (4). By contrast, GrB has an unusual preference for cleaving after Asp and is thought to act like an apical caspase in triggering classical apoptosis (2). The role of GrB in cell-mediated immunity is supported by the phenotype of GrBdeficient mice, which produce CLs that cannot induce rapid DNA degradation in target cells, although killing still occurs, possibly via other granzymes (5). Furthermore, apoptosis of transplanted allogeneic hepatocytes in rats is reduced by treatment with GrB inhibitors (6), elevated GrB serum levels occur in several diseases including rheumatoid arthritis, and GrB is implicated in the generation of autoimmune antigens (7).The importance of the granule pathway to CL function is illustra...

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Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins

et al. 2010

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Killer lymphocytes induce apoptosis by the release of cytotoxic mediators from specialized secretory lysosomes, called cytotoxic granules, into the immunological synapse formed with a cell targeted for elimination. Methods are presented here for isolating CTL and NK cell cytotoxic granules using cell disruption by nitrogen cavitation followed by continuous Percoll density gradient fractionation. Protocols are also given for purifying the key cytolytic molecules (perforin, granzyme A, granzyme B, and granulysin) from isolated cytotoxic granules by fast protein liquid chromatography. Curr. Protoc. Cell Biol. 47:3.37.1‐3.37.29. © 2010 by John Wiley & Sons, Inc.

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Expression and Purification of Recombinant Human Granzyme B from Pichia pastoris

Cited by 58 publications

References 25 publications

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Mouse granzyme A induces a novel death with writhing morphology that is mechanistically distinct from granzyme B-induced apoptosis

Granzyme B Encoded by the Commonly Occurring Human RAH Allele Retains Pro-apoptotic Activity

Isolation of Cytotoxic T Cell and NK Granules and Purification of Their Effector Proteins

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