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 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.

scholarly journals MazG, a Nucleoside Triphosphate Pyrophosphohydrolase, Interacts with Era, an Essential GTPase in Escherichia coli

2002 ◽  
Vol 184 (19) ◽  
pp. 5323-5329 ◽  
Author(s):  
Junjie Zhang ◽  
Masayori Inouye
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Kanamycin Resistance ◽  
Nucleoside Triphosphate ◽  
Cellular Functions ◽  
Genomic Libraries ◽  
E Coli ◽  
Yeast Two Hybrid System ◽  
Nucleoside Triphosphate Pyrophosphohydrolase

ABSTRACT Era is an essential GTPase in Escherichia coli, and Era has been implicated in a number of cellular functions. Homologues of Era have been identified in various bacteria and some eukaryotes. Using the era gene as bait in the yeast two-hybrid system to screen E. coli genomic libraries, we discovered that Era interacts with MazG, a protein of unknown function which is highly conserved among bacteria. The direct interaction between Era and MazG was also confirmed in vitro, being stronger in the presence of GDP than in the presence of GTPγS. MazG was characterized as a nucleoside triphosphate pyrophosphohydrolase which can hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi, with a preference for deoxynucleotides. A mazG deletion strain of E. coli was constructed by replacing the mazG gene with a kanamycin resistance gene. Unlike mutT, a gene for another conserved nucleotide triphosphate pyrophosphohydrolase that functions as a mutator gene, the mazG deletion did not result in a mutator phenotype in E. coli.

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

scholarly journals Identification, Bioinformatics Analyses, and Expression of Immunoreactive Antigens of Mycoplasma haemofelis

2011 ◽  
Vol 18 (8) ◽  
pp. 1275-1281 ◽  
Author(s):  
Joanne B. Messick ◽  
Andrea P. Santos
Keyword(s):  
Expression System ◽  
Cellular Functions ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Genomic Expression ◽  
Recombinant Fusion Proteins ◽  
Specific Pathogen ◽  
Immunogenic Proteins ◽  
High Level

ABSTRACTMycoplasma haemofelisinfection frequently causes anemia in cats. Despite an intense immune response and/or antibiotic treatment, cats often remain asymptomatic carriers following infection. Our hypothesis is that detection of antibodies toM. haemofelisis a sensitive approach for identifying infected cats, particularly carriers. To date, no immunoassay has been developed. This is due largely to the inability to cultureM. haemofelis in vitro; hence, a source of antigen is not readily available. The objective of this study was to identify, express, and purify immunogenic proteins ofM. haemofelis. To accomplish this, two whole-genomic expression libraries were created in the Lambda ZapII vector and immunoscreened with preimmune plasma, plasma from specific-pathogen-free cats, and pooled acute- and convalescent-phase plasma from experimentally infected cats. The inserts from 21 immunoreactive clones were sequenced, resulting in the identification of 60 genes coding for putative proteins necessary for diverse cellular functions, along with several novel genes ofM. haemofelis. Fragments of selected genes based on bioinformatic analyses were PCR amplified, cloned into a high-level protein expression system, and subsequently expressed inEscherichia colias a His6-fusion protein. The recombinant fusion proteins ofM. haemofeliswere purified and evaluated as an antigen in a Western blot to verify the findings of previous immunoscreening. Together with bioinformatics analyses of individual genes, this approach provided several putative candidate antigens. Five antigens ofM. haemofeliswere reactive by Western blotting against the immune plasma and negative against nonimmune plasma; these antigens might be useful serologic or even vaccine targets.

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

Effect of rhBMP-2 produced by Escherichia coli expression system on bone formation in rat calvarial defects

2009 ◽  
Vol 39 (1) ◽  
pp. 77
Author(s):  
Suk-Hoon Kwon ◽  
Hyun-Chang Lim ◽  
Kyung-Hee Choi ◽  
Min-Soo Kim ◽  
Ji-Hyun Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bone Formation ◽  
Expression System ◽  
Calvarial Defects ◽  
Escherichia Coli Expression

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

Sign in / Sign up

Export Citation Format

Share Document