High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells

Yang, Wei; Xia, Weiliang; Mao, Jingjing; Xu, Danning; Chen, Jianhe; Feng, Shan; Wang, Jianhua; Li, Hua; Theisen, Claus Friis; Petersen, Jørn Meidahl; Thórólfsson, Matthı́as; Rasmussen, Hanne B.; Junker, Flemming; Boel, Esper; Su, Jing

doi:10.1016/j.pep.2010.09.001

Cited by 12 publications

(5 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After 4 h at 37 °C incubation, the activation level of CatC reached 9.6 U/mg, corresponding to approximately 23 mg of mature recombinant CatC per liter of culture ( Figure 6). This enzymatic activity toward the studied synthetic substrate was high and comparable with that of the results reported previously (Dahl et al, 2001;Yang et al, 2011).…”

Section: Expression and Activation Of Recombinant Human Catcsupporting

confidence: 91%

“…Several expression systems were studied in recent years for the recombinant production of human CatC. In those previous reports, recombinant production of CatC was described in mammalian and baculovirus-infected insect cell systems (Dahl et al, 2001;Yang et al, 2011). However, mammalian systems are complicated, expensive to handle, and mostly inappropriate for high-level expression of a functional enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…To date, a number of studies have been reported on recombinant expression of human CatC that were performed in insect and mammalian cell systems (Dahl et al, 2001;Yang et al, 2011). However, these systems are high-cost methods that require complex and expensive media and involve cumbersome work procedures.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Daglioglu¹

2017

Turk J Biol

View full text Add to dashboard Cite

The yeast Pichia pastoris expression system was investigated for the production of human cathepsin C (CatC) recombinant protein. The full-length CatC cDNA, corresponding to amino acids 12-475, was synthesized from interleukin-2 (IL-2) stimulated human peripheral blood mononuclear cells and subcloned in the pGEM-T cloning vector. After confirming the DNA sequence of the insert, the gene was cloned into the pPICZαA expression vector under the control of the methanol-inducible alcohol oxidase (AOX1) promoter and transformed to P. pastoris X-33 cells. The expressed protein was secreted into the culture medium through the α-factor mating signal sequence of the expression vector. Analysis of the culture supernatant revealed that the recombinant human CatC was secreted as a 58-kDa molecule, indicating that human CatC was accumulated in the culture supernatant as proform composed of the residual propart, the activation peptide, and the heavy and light chains. Extracellular recombinant proCatC was further activated by cysteine endoprotease papain in vitro and its activity was confirmed by assays using a synthetic substrate.

show abstract

Section: Expression and Activation Of Recombinant Human Catcsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Daglioglu¹

2017

Turk J Biol

View full text Add to dashboard Cite

show abstract

“…Besides its application in the expression of recombinant protein therapeutics, cell line development technologies are also critical in the expression of enzymes like dipeptidyl peptidase I (DPPI) which may expedite the industrial use of the peptidase as a processing enzyme [117]. Interestingly, these technologies may also be utilized for discovery and characterization of proteins that may be potential drug targets.…”

Section: Discussionmentioning

confidence: 99%

Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

2013

View full text Add to dashboard Cite

From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics.

show abstract

“…12A) (48). In the comparatively well-studied mammalian cathepsin C, it is known that the first three domains are removed in a maturation process, to generate a catalytically active heavyϩlight chain (HL) species (49). In order to detect potential processing intermediates of T. thermophila Cth4p, we expressed a copy that The processed Grl products retain N-terminal extensions in ⌬cth4 cells.…”

Section: Figmentioning

confidence: 99%

Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

2015

View full text Add to dashboard Cite

In many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila, efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 (CTH3). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4 results in cells (⌬cth4) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, ⌬cth4 cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the ⌬cth4 defects by mutating conserved catalytic residues, indicating that the in vivo function of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The ⌬cth4 defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates. R egulated secretion of peptides from intracellular stores plays many key roles in intercellular communication and tissue coordination in animals (1). A highly specialized organelle in neuroendocrine cells, the secretory granule, is required for peptide generation, storage, and release (2). The bioactive peptides are generated during secretory granule formation by the action of proteolytic enzymes on polypeptide precursors (3). Proteolytic processing occurs in a post-trans-Golgi network (TGN) compartment, the immature granule, to which propeptides are sorted away from bulk protein traffic, and is a multistep process involving several classes of enzymes (4-6). The best studied among these are a family of serine proteases called prohormone convertases (7). Related to bacterial subtilisins, prohormone convertases (PCs) catalyze endoproteolytic cleavage at defined motifs in their substrates. The products of that cleavage can then undergo trimming, for which one important factor is carboxypeptidase E (CPE) (8). Defects in both PCs and CPE are linked with disease (9, 10).Signaling via peptide secretion appears to be widespread in eukaryotes and has also been well documented for fungi, amoebozoa, and plants(11-13). In the oligohymenophorean ciliates Tetrahymena thermophila and Paramecium tetraurelia, regulated peptide secretion occurs from organelles that share striking similarities with secretory granules in animals (14, 15), although...

show abstract

High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells

Cited by 12 publications

References 37 publications

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

Secretion of Polypeptide Crystals from Tetrahymena thermophila Secretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

Contact Info

Product

Resources

About