Refolding, Purification, and Characterization of Human Recombinant PDE4A Constructs Expressed in Escherichia coli

Richter, Wito; Hermsdorf, Thomas; Lilie, Hauke; Egerland, Ute; Rudolph, Rainer; Kronbach, Thomas; Dettmer, D

doi:10.1006/prep.2000.1260

Cited by 14 publications

(5 citation statements)

References 18 publications

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“…Previous studies, including our own, showed by gel filtration and density gradient centrifugation that the catalytic domains of PDE4A and PDE4B behave as oligomers (40,(42)(43)(44). As the catalytic domains of the PDE4 subtypes are highly conserved, it is unlikely that this discrepancy is due to sequence differences between PDE4A/B and the PDE4D used in our present study.…”

Section: Table I Physicochemical Properties Of the Pde4d Constructscontrasting

confidence: 39%

“…Anomalous behavior of PDE4 also has been observed by SDS-PAGE with the protein migrating with an apparent molecular weight higher than the theoretical molecular weight. In addition, several other reports have attempted to investigate the state of oligomerization of PDE4 with mixed results due to the extensive aggregation experienced with purified recombinant proteins (40,42).…”

Section: Table I Physicochemical Properties Of the Pde4d Constructsmentioning

confidence: 99%

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Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Richter

Conti

2002

Journal of Biological Chemistry

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The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.The second messengers cAMP and cGMP are key molecules for transducing the action of extracellular signals such as hormones, neurotransmitters, and light into the most diverse cell functions, thus playing an important role in a wide array of physiological processes that include vision, cell growth and division, memory, and immune response (1-5). Intracellular cyclic nucleotide levels are determined by the rates of their synthesis by adenylyl cyclases and of their degradation through phosphodiesterases, enzymes that hydrolyze the phosphodiester bond and generate the corresponding 5Ј-nucleoside monophosphates.Cyclic nucleotide phosphodiesterases (PDEs) 1 compose a superfamily of isoenzymes that are divided into 11 PDE families on the basis of their sequence homology and enzymatic properties (2). They all share a highly conserved catalytic domain of about 270 amino acids fused to additional N-and/or C-terminal sequences that contain distinct domains unique to the members of a PDE family. These terminal domains determine several properties of the PDEs such as the regulation of enzyme activity by post-translational modifications (e.g. phosphorylation by PKA, PKB, ERK2, CaMK, and PKG, Refs. 6 -10) and binding of other messenger molecules (e.g. cGMP, Ca 2ϩ -calmodulin, and phosphatidic acid, Refs. 11-13), or by specifying the subcellular localization of the enzymes by protein-proteininteractions and membrane insertion (14 -16). The terminal domains of the PDEs, therefore, provide diverse modules for coordinating PDE activity with the overall signaling network specific to a cel...

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Section: Table I Physicochemical Properties Of the Pde4d Constructscontrasting

confidence: 39%

Section: Table I Physicochemical Properties Of the Pde4d Constructsmentioning

confidence: 99%

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Richter

Conti

2002

Journal of Biological Chemistry

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“…50 In contrast, presence of residual proteases in the DS can significantly influence the long-term storage stability depending on their level in the preparations. 51 Rapid protease-catalyzed hydrolysis was observed for affinity-purified rhTPO at pH 6-8, 34 and a Chinese hamster ovary-derived high-purity human IgG1 mAb. 35 The carboxypeptidase-catalyzed C-terminal lysine clipping in mAb's leads to charge heterogeneity, which may potentially impact the protein stability, as the number of charges in mAb's plays a role in protein aggregation.…”

Section: Host Cell Proteinsmentioning

confidence: 98%

“…In contrast, presence of residual proteases in the DS can significantly influence the long‐term storage stability depending on their level in the preparations . Rapid protease‐catalyzed hydrolysis was observed for affinity‐purified rhTPO at pH 6–8, and a Chinese hamster ovary‐derived high‐purity human IgG1 mAb .…”

Section: Drug Substance Manufacturingmentioning

confidence: 99%

Impact of Residual Impurities and Contaminants on Protein Stability

Wang

Arunkumar

Thakkar

2014

Journal of Pharmaceutical Sciences

100

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“…7 In most cases, the (His) 6 affinity tag does not interfere with the structure and function of the purified protein. However, in some reported cases, the position of the (His) 6 tag on the recombinant protein influences its accessibility for purification 8 or reduces its interaction with its ligand 9 (DNA, RNA, antigen, etc.). For this reason, it was decided to make in parallel two expression constructs, whereby the (His) 6 tag was placed on either the N-or C-terminus of the KgmB protein.…”

Section: Introductionmentioning

confidence: 99%

Different expression levels of two KgmB-His fusion proteins

Marković¹,

Vojnović²,

Jovanovic³

et al. 2005

JSCS

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The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

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Refolding, Purification, and Characterization of Human Recombinant PDE4A Constructs Expressed in Escherichia coli

Cited by 14 publications

References 18 publications

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Dimerization of the Type 4 cAMP-specific Phosphodiesterases Is Mediated by the Upstream Conserved Regions (UCRs)

Impact of Residual Impurities and Contaminants on Protein Stability

Different expression levels of two KgmB-His fusion proteins

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