E. coli expression system

Ranibizumab is produced in E. coli expression system in a nutrient medium containing the tetracycline antibiotic. It has a molecular weight of 48 kDa. 

The E. coli expression system used is based on the pQE-80L expression system. Sequences inserted into the multiple cloning site can be expressed as native proteins bearing an N-terminal hexahistidine tag upon IPTG induction of the T5 promoter in suitably transformed E. coli cells. 

When using common E. coli expression system, recombinant allergens are present, either soluble in the supernatant of the cell lysate, or the proteins aggregate and form poorly soluble inclusion bodies. Inclusion bodies have to be solubilized under denaturing conditions (e.g., urea, guanidine-HCl treatment) and the protein has to be carefully refolded (Rea et al. 2004). One advantage of a eukaryotic yeast expression system is that yeast cells can segregate the expressed protein into the culture medium and thus can easily be purified. 

In an Escherichia coli expression system for the aqualysin I precursor, the precursor is processed autoproteolytically into the mature 28-kDa enzyme by treatment at 65 ° C.23) In this case, the N-terminal pro-sequence is required for the production of active enzyme and functions to stabilize the precursor structure.283 The C-terminal pro-sequence is not essential for the production of active aqualysin 1,293 but seems to be involved in the translocation of the precursor across the cytoplasmic membrane.303 In a Thermus thermophilus expression system,313 the C-terminal pro-sequence is required for the production and extracellular secretion of active aqualysin I.323 In an E. coli expression system for the subtilisin E gene, the N-terminal pro-sequence is essential for the production of active enzyme,333 as in the case of aqualysin I. The requirement of the pro-sequence is also shown in vitro for the refolding of the inactive mature protein to an active enzyme.34 353 The functions of the N-terminal pro-sequences of aqualysin I and subtilisin E seem to be similar. 

In general, the production of good MAbs depends to a large part upon the quality, purity and availability of antigen. In the production of the anti-RANTES chemokine monoclonal antibodies described here, a pET-3 E. coli expression system was used that allowed the manufacture of large quantities of recombinant human RANTES protein (see Notes 1,2, and 3). The recombinant protein was also used as a positive control in Western and immunoprecipitation experiments performed at later stages of the characterization of the MAbs (1). 

This circumstance made our stated aim of comparing dynamic behavior of the free protein with the bound protein more difficult. We developed a streamlined production method that takes advantage of the differential expression levels of p50 and p65 in the E. coli expression system. Using this... 

One benefit of the simplicity of the E. coli expression system is the ability to test dozens or hundreds of variables in parallel in small volumes (0.5-10 ml)51,52 or even directly in colonies on a plate (see Section 9.19.5.6).53-55 This allows the assessment of the relative utility of constructs, fusion tags, growth and induction conditions,... 

The E coli expression system described here has also proven useful for purifying plant ALS enzymes. Because it is present in such small amounts, ALS has been difficult to purify from plant sources. Purifying the plant enzymes expressed in bacteria has provided material for use in enzymatic and structural studies as well as for the generation of immunological reagents. 

USA interferon alfa-2b interferon I ntron A Alfatronol, Depo Interferon alpha, Viraferon Recombinant IFN-alpha2b from the clone Hif-SN206 with substitution at 23 (Arg) and 34 (His) from an E. coli expression system Cancer, hepatitis... 

Switzerland peginterferon alfa-2a interferon Pegasys Recombinant interferon alfa-2a from an E. coli expression system linked to polyethylene glycol Hepatitis... 

USA human insulin Zn suspension insulin Humulin L Humulin Lente A mixture of crystalline and amorphous recombinant human insulin obtained from an E. coli expression system Diabetes... 

E. coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes... 

To aid in purification of a eukaryotic protein produced in an E. coli expression system, researchers often modify the cDNA encoding the recombinant protein to facilitate its separation from endogenous E. coli proteins. A commonly used modification of this type is to add a short nucleotide sequence to the end of the cDNA, so that the expressed protein will have six histidine residues at the C-termlnus. Proteins modified in this way bind tightly to an affinity matrix that contains chelated nickel atoms, whereas most E. cofi proteins will not bind to such a matrix. The bound proteins can be released from the nickel atoms by decreasing the pH of the surrounding medium. In most cases, this procedure yields a pure recombinant protein that is functional, since addition of short amino acid sequences to either the C-terminus or the N-terminus of a protein usually does not Interfere with the protein s biochemical activity. 

The P. past oris expression system produced high yields of secreted protein. The recombinant protein represents the majority of the protein supernatant without further purification. The E. coli expression system was found to be unsuitable for expression of soluble and active ferulic acid esterase from a fungal source. 

In this section, we describe methods to produce 100 mg quantities of a soluble recombinant variant of human TPST-1 by using an E. coli expression system. Expression of this TPST-1 variant in E. coli results in incorrectly folded protein that accumulates in inclusion bodies. Active enzyme is obtained by solubilization of inclusion bodies followed by functional refolding of TPST-1. 

Moreover, the E.coli expression systems do not allow direct study of light activated chloroplast promoters and sequences. 

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