Biomaterial Database

One-step purification of insoluble hydantoinase overproduced in Escherichia coli. 2007

Chung-Jen Chiang, and Hong-Chen Chen, and Yun-Peng Chao, and Jason T C Tzen

Department of Life Science, National Chung-Hsing University, Taichung, Taiwan.

Over-expression of hydantoinase from Agrobacterium radiobacter NRRL B11291 (HDTar) results in the formation of insoluble aggregates in Escherichia coli. As previously reported, recombinant HDTar could be obtained in a homogeneous form using one chromatographic step. However, soluble proteins are required for the pre-treatment in several steps before proceeding to the chromatographic purification step. In this study, we reported a method based on artificial oil bodies (AOBs) to obtain homologous HDTar from its insoluble form in one step. By linkage of HDTar to intein-oleosin gene fusion, the tripartite fusion protein was over-expressed as aggregates in E. coli. Upon sonication, the mixture comprising plant oil and the insoluble fusion protein was readily assembled into AOBs. Further induction for peptide cleavage mediated by intein, the bound HDTar was liberated from AOBs, and the protein free of fusion tags was then recovered. As a result, refolded HDTar was amplified by over 300-fold. Obviously, this simplified method provides an efficient way to obtain HDTar with high yield and high purity.

UI	MeSH Term	Description	Entries
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D004591	Electrophoresis, Polyacrylamide Gel	Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.	Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000581	Amidohydrolases	Any member of the class of enzymes that catalyze the cleavage of amide bonds and result in the addition of water to the resulting molecules.	Amidases,Amidohydrolase
D016133	Polymerase Chain Reaction	In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.	Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain
D050939	Gene Fusion	The GENETIC RECOMBINATION of the parts of two or more GENES resulting in a gene with different or additional regulatory regions, or a new chimeric gene product. ONCOGENE FUSION includes an ONCOGENE as at least one of the fusion partners and such gene fusions are often detected in neoplastic cells and are transcribed into ONCOGENE FUSION PROTEINS. ARTIFICIAL GENE FUSION is carried out in vitro by RECOMBINANT DNA technology.	Fusion, Gene,Fusions, Gene,Gene Fusions
D017931	DNA Primers	Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.	DNA Primer,Oligodeoxyribonucleotide Primer,Oligodeoxyribonucleotide Primers,Oligonucleotide Primer,Oligonucleotide Primers,Primer, DNA,Primer, Oligodeoxyribonucleotide,Primer, Oligonucleotide,Primers, DNA,Primers, Oligodeoxyribonucleotide,Primers, Oligonucleotide