Abstract
Cytosolic carboxypeptidase 6 (CCP6) is a member of cytosolic carboxypeptidase (CCP) family that catalyze the removal of polyglutamate side chains from protein substrates. Biochemical and biophysical characterization of CCPs requires large quantities of purified proteins. However, no method describing the expression of any mammalian CCP family member from bacteria has been published to our best knowledge. After considerable efforts to improve the solubility of mammalian CCPs expressed in bacteria, including the optimization of induction temperature and by using different receptive cells, we were able to get less expression of mouse CCP6 in soluble fraction of bacterial lysates. We report in this article, the bacterial expression of CCP6 using Arctic Express (DE3) competent cells that co-express the chaperonin system GroEL and GroES from Oleispira antarctica. However, to achieve a large number of soluble target proteins, the expression conditions still need to be further optimized.
Highlights
Introduction their functions remain unknownCurrently, studying the biochemistry of eukaryotic CCPs depends on cell orIn mammals, cytosolic carboxypeptidase family catalyzes the demodification of a unique posttranslational modification-polyglutamylation
We found that mouse original Cytosolic carboxypeptidase 6 (CCP6) existed largely as inclusion bodies when expressed in prokaryotic cells
The plasmids of HT-CCP6-45 and HT-CCP6-167 were transformed into Rosetta (DE3) competent cells, respectively
Summary
Studying the biochemistry of eukaryotic CCPs depends on cell or. Cytosolic carboxypeptidase family catalyzes the demodification of a unique posttranslational modification-polyglutamylation. Polyglutamylation gives rise to variable lengths of glutamate side chains on the γ-carboxyl groups of glutamic acid residues in the insect cell expression systems, which are time-consuming and expensive. To date there are no published methods describing the expression and purification of any mammalian CCP family member from bacteria. In the process of this modification, free glutamate residues are initially condensed to the γ-carboxyl group of a glutamate in the primary sequence of proteins to form a branched chain. Additional glutamates are linked to this branching point glutamate by peptide bond (α-carboxyl) to form polyglutamylation side chains of different lengths (Figure 1). Its initiation and elongation are catalyzed by the tubulin tyrosine ligase like (TTLL)
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