scholarly journals Mapping residues of SUMO precursors essential in differential maturation by SUMO-specific protease, SENP1

2005 ◽  
Vol 386 (2) ◽  
pp. 325-330 ◽  
Author(s):  
Zheng XU ◽  
Shannon W. N. AU
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Tissue Distribution ◽  
Catalytic Domain ◽  
Expression System ◽  
Proteolytic Processing ◽  
Maturation Process ◽  
Specific Protease ◽  
Escherichia Coli Expression

SUMO (small ubiquitin-related modifier) is a member of the ubiquitin-like protein family that regulates cellular function of a variety of target proteins. SUMO proteins are expressed as their precursor forms. Cleavage of the residues after the ‘GG’ region of these precursors by SUMO-specific proteases in maturation is a prerequisite for subsequent sumoylation. To understand further this proteolytic processing, we expressed and purified SENP1 (sentrin-specific protease 1), one of the SUMO-specific proteases, using an Escherichia coli expression system. We show that SENP1 is capable of processing all SUMO-1, -2 and -3 in vitro; however, the proteolytic efficiency of SUMO-1 is the highest followed by SUMO-2 and -3. We demonstrate further that the catalytic domain of SENP1 (SENP1C) alone can determine the substrate specificity towards SUMO-1, -2 and -3. Replacement of the C-terminal fragments after the ‘GG’ region of SUMO-1 and -2 precursors with that of the SUMO-3, indicates that the C-terminal fragment is essential for efficient maturation. In mutagenesis analysis, we further map two residues immediately after the ‘GG’ region, which determine the differential maturation. Distinct patterns of tissue distribution of SENP1, SUMO-1, -2 and -3 are characterized. Taken together, we suggest that the observed differential maturation process has its physiological significance in the regulation of the sumoylation pathway.

Expression of adenylate kinase fused mouse ubiquitin active enzyme in Escherichia coli and its application in ubiquitination

2021 ◽  
Author(s):  
Xiaoliang Liu ◽  
Ling Hu ◽  
Yuan Zhang ◽  
Hongtao Li
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Chemical Synthesis ◽  
Adenylate Kinase ◽  
Energy Supply ◽  
Expression System ◽  
Synthesis Process ◽  
Cellular Functions ◽  
Escherichia Coli Expression

Abstract Ubiquitination, is involved in the regulation of numerous cellular functions. Researches in the ubiquitin realm rely heavily on ubiquitination assays in vitro and require large amounts of ubiquitin-activating enzyme (UBA1) and keep ATP supplies. But UBA1 is hard to be obtained with large quantities using reported methods. We fused Escherichia coli adenylate kinase (adk) and mouse UBA1 obtained fusion protein adk-mUBA1. The expression level of adk-mUBA1 increased about 8-fold than that of mUBA1 in Escherichia coli expression system, and adk-mUBA1 was easily purified to 90% purity via two purification steps. The purified adk-mUBA1 protein was functional for ubiquitination and could use ATP in addition to ADP as energy supply and had a higher catalytic activity than mUBA1 in cell lysis. Adk-mUBA1 can be applied to preparing ubiquitin modified substrates and kinds of ubiquitin chains in chemical synthesis process and is preferable application than mUBA1 in vitro ubiquitination.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

scholarly journals Desumoylation Activity of Axam, a Novel Axin-Binding Protein, Is Involved in Downregulation of β-Catenin

2002 ◽  
Vol 22 (11) ◽  
pp. 3803-3819 ◽  
Author(s):  
Takayuki Kadoya ◽  
Hideki Yamamoto ◽  
Toshiaki Suzuki ◽  
Akira Yukita ◽  
Akimasa Fukui ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Signaling Pathway ◽  
Catalytic Domain ◽  
Binding Protein ◽  
Wnt Signaling Pathway ◽  
Binding Domain ◽  
Axis Formation ◽  
Intact Cells ◽  
Specific Protease

ABSTRACT Axam has been identified as a novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of β-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme). Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of β-catenin. An Axam fragment which contains both domains was able to decrease the level of β-catenin. On substitution of Ser for Cys547 in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate β-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, AxamC547S showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate β-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.

scholarly journals Construction of an antibody Fv-Epo receptor chimera extracellular domainusing Escherichia coli expression system

2000 ◽  
Vol 40 (supplement) ◽  
pp. S186
Author(s):  
Takeshi Kitamura ◽  
Kouhei Tsumoto ◽  
Masahiro Kawahara ◽  
Hiroshi Ueda ◽  
Teruyuki Nagamune ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Epo Receptor ◽  
Escherichia Coli Expression

Production of recombinant IGF1 and its action on neuroblastoma cells in vitro

2018 ◽  
Vol 18 (4) ◽  
pp. 34-41
Author(s):  
Sergey A. Ishuk ◽  
Elena G. Bogomolova ◽  
Olga A. Dobrovolskaya ◽  
Alyona O. Akhmetshina ◽  
Daria S. Krasnoshchek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Cation Exchange ◽  
Expression System ◽  
Neuroblastoma Cells ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Recombinant Insulin ◽  
Prokaryotic Expression System

This study aimed to develop a method for producing human recombinant insulin-like growth factor (IGF-1) based on a prokaryotic expression system and to characterize the highly purified protein. To achieve the study’s goal, the following methods were conducted: we performed automated chemical synthesis of DNA, constructed the expression plasmid, obtained Escherichia coli cell-producers of human recombinant IGF-1, cultivated the obtained producer cells with the induction of recombinant protein synthesis by isopropyl-β-D-1-thiogalactopyranoside and lactose, and purified human recombinant IGF-1 with affinity and cation exchange chromatography. The recombinant protein IGF-1 forms inclusion bodies during synthesis in Escherichia coli BL21 cells that contain plasmid pET28-IGF-1. Purified recombinant protein was obtained with a purity of 98% using affinity and cation exchange chromatography methods. The protein yield was 6 mg of human recombinant IGF-1 from 1 g of raw biomass. The resulting protein has the ability to protect Neuro 2a neuroblastoma cells from death caused by the deprivation of serum in the culture medium and can stimulate the differentiation of cells into neurons. Thus, a highly purified human recombinant IGF-1 was obtained. This protein has biological activity and is suitable for preclinical studies.

Cotranslational Incorporation of a Structurally Diverse Series of Proline Analogues in an Escherichia coli Expression System

ChemBioChem ◽  
2004 ◽  
Vol 5 (7) ◽  
pp. 928-936 ◽  
Author(s):  
Wookhyun Kim ◽  
Anna George ◽  
Melissa Evans ◽  
Vincent P. Conticello
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Escherichia Coli Expression
Sign in / Sign up

Export Citation Format

Share Document