Cloning and expression of functional rabbit muscle creatine kinase in Escherichia coli. Addressing the problem of microheterogeneity.
نویسندگان
چکیده
The gene encoding rabbit muscle creatine kinase (CK) has been subcloned into a single plasmid, and the protein expressed in a soluble and functional form in Escherichia coli. The amino terminus, specific activity, and electrophoretic mobility of the E. coli-expressed creatine kinase are all identical with that of creatine kinase purified from rabbit skeletal muscle. Surprisingly, isoelectric focusing shows that the expressed protein displays no less heterogeneity than the tissue-purified material. The identification of the source(s) of this heterogeneity is important for the preparation of highly homogeneous material needed for structural studies and clinical applications. This issue also has implications for studies of the developmental regulation and tissue localization of the various CK genes. Our results allow us to eliminate some of the proposals, such as the presence of multiple alleles, alternative ribosomal initiation sites, and post-translational glycosylation or phosphorylation that have been suggested to explain the presence of the numerous isoforms present in apparently pure preparations of CK.
منابع مشابه
Cloning and Expression of the Coat Protein Gene of Barley Yellow Dwarf Virus-PAV in Escherichia coli
متن کامل
Cloning and optimization of phytase enzyme gene expression in Escherichia coli
Introduction Phytase is an enzyme that has the ability to break down phytic acid into myoinositol and mineral phosphate, and widely uses as an additive in animal foods. The aim of this study was to achieve a high level of bacterial phytase expression in PET26b expression host. Materials and Methods To generate the recombinant phytase enzyme, the target gene was introduced into the expression ...
متن کاملExpression Cloning of Recombinant Escherichia coli lacZ Genes Encoding Cytoplasmic and Nuclear P-galactosidase Variants
Objective(s) Nonviral vector can be an attractive alternative to gene delivery in experimental study. In spite of some advantages in comparison with the viral vectors, there are still some limitations for efficiency of gene delivery in nonviral vectors. To determine the effective expression, the recombinant Escherichia coli lacZ genes were cloned into the different variants of pcDNA3.1 and the...
متن کاملCloning and sequencing of ompf Salmonella typhi Salmonella ompf gene in Escherichia coli Origami
Background and Aim: Salmonella Typhi belongs to the family Enterobacteriaceae, gram-negative bacilli and causes gastrointestinal diseases such as typhoid. This bacterium has a special structure and various genes, including the ompf gene (outer membrane protein). Recent studies have shown the possibility of using ompf in the development of a diagnostic tuberculosis vaccine. Therefore, the aim of...
متن کاملCloning and evaluation of gene expression and purification of gene encoding recombinant protein containing binding subunit of coli surface antigens CS1 and CS2 from Enterotoxigenic Escherichia coli
Background & Objective: Enterotoxigenic Escherichia coli (ETEC) is a major causative agent of diarrhea. Enterotoxins and the colonization factors (CFs) are major virulence factors in ETEC infections. The bacterium binds to the intestinal epithelial cell surface through colonization factors and produces enterotoxins that cause excessive fluid and electrolyte secretion in the lumen of the intesti...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
عنوان ژورنال:
- The Journal of biological chemistry
دوره 266 18 شماره
صفحات -
تاریخ انتشار 1991