Recombinant over-expressed proteins

It is clear that in this brief overview of molecular biology, we have not covered a number of important areas that have an important impact on the study of metalloproteins. These include molecular cloning and recombinant DNA technology, which allow proteins to be over-expressed and individual amino acids to be mutated to any other of the 19 protein amino acids genome and proteome analysis that enables the sequences of all the genes of the entire organisms to be determined, and the quantification, localization, interactions and, where possible, activities and identification of all of the proteins in an organism,... 

The host bacteria used for production of recombinant proteins are usually E. coli, or Bacillus subtilis they may express proteins at 1 % to over 50% of the cellular protein, depending on such variables as the source, promoter structure, and vector type. Generally the proteins are expressed intracellularly, but leader sequences for excretion may be included. In the latter case, the protein is generally excreted into the periplasmic space, which limits the amount that can be produced. Excretion from grampositive species such as B. subtilis sends the product into the culture medium, with little feedback limitation on total expression level. 

Over several decades, multiple vector systems for recombinant gene expression in E. coli have been developed. Modem vectors suitable for recombinant protein production vary in the used promoter system in the presence or absence of coding sequences for affinity tags upstream or downstream of the multiple cloning site (MCS) and of sequences coding for leader peptides for the protein export. Moreover, different origins of replication (ori), antibiotic selection marker genes and MCS are used. 

Recombinant protein over-expressed in soluble form in the cytoplasm E. coli bacteria... 

Over-expression in E. coli. The reading frame of fae-1 was expressed in E. coli using the pET expression system. Various temperatures and IPTG concentrations were tested to find the best conditions in order to produce the largest quantity of soluble protein. However, the recombinant feruloyl esterase proved to be insoluble (Figure 3) and non-active irrespective of the conditions employed. 

Live cells, which overexpress the protein/glycoprotein of interest, can form excellent targets for antibody selection when grown either in suspension or as adherent monolayers in multiwell plates. For example, tumour cells that over-express growth factor receptors, or Chinese hamster ovary cells expressing recombinant human proteins, or insect cells expressing baculovirus constructs. 

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