Recombinant protein expression in E.coli

Baneyx, F. 1999. Recombinant protein expression in E. coli. Current Opinion in Biotechnology 10, 411-421. 

Obviously, phage display technology requires the protein to be properly folded and stable as a fusion with the coat protein. Successful display may depend on the bacterial host and on growth conditions. General strategies to improve recombinant protein expression in E.coli [36] should be transposable to the phage display context. 

Kundu et al.64 used MEKC conditions to assess the purity of two recombinant proteins a cytomegalovirus-CMP-KDO synthetase fusion protein expressed in E. coli and a hepatitis C viral protein expressed in CHO cells. Proteins were prepared in a 10-mM Tris-1% SDS buffer (pH 8.5) and analyzed in a 10-mM borate-100-mM SDS buffer (pH 9.5) in uncoated capillaries. The level of impurities, which varied with the method of protein production, agreed within 5% with results obtained by densitometric scanning of SDS-PAGE gels of the same materials. 

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. 

This report describes the presence of significant amounts of e-N-acetyllysine in rpST and rbST, eukaryotic proteins expressed in a prokaryotic system. Initial work from our laboratory has also demonstrated the presence of this modified amino acid in two other recombinant eukaryotic proteins expressed in E. coli, bovine placental lactogen and human tissue factor pathway inhibitor (16). ESMS, amino acid sequencing and amino acid analyses were utilized to demonstrate the presence of e-N-acetyllysine in these two recombinant proteins. These data established that this modified amino acid is present in several distinct recombinant eukaryotic proteins expressed in E. coli. 

Similarly, PrP extracted from hamster brains into 10 mM aqueous sodium phosphate solution at pH 7.5 yielded CD and infrared spectra that are typical for a-helical proteins (Pan et ai, 1993). Overall, the optical spectra thus indicate that natural PrP, with its numerous post-translational modifications has a similar conformation as recombinant PrP expressed in E. coli and dissolved in nondenaturing aqueous solvents. On a different line, evidence has also been presented that neither the C-terminal GPI-anchor nor the carbohydrate moieties in the positions 181 and 197 (Fig. 1) are essential molecular components with regard to the transformation of PrP into forms that are associated with prion infectivity (Stahl et al, 1987). These data clearly emphasize the relevancy of structural studies with recombinant prion proteins for deeper insights into structure and function of natural prion proteins. 

The gene of Aequorea GFP was cloned by Prasher et al. (1992), and expressed in E. coli and Caenorhabditis elegans by Chalfie et al. (1994) and in E. coli by Inouye and Tsuji (1994a). The X-ray structure of recombinant GFP was solved by Ormo et al. (1996) and Yang et al. (1996,1997). The protein is in the shape of a cylinder consisting of 11 strands of (3-sheets and an a-helix inside (which contains the chromophore), with short helical segments on the ends of the cylinder. Thus the chromophore is sealed and protected from the outside medium. 

The cDNA for this photoprotein has been cloned and expressed in E. coli, and the recombinant protein obtained was named mitrocomin (Fagan et al., 1993). Mitrocomin consists of 190 amino acid residues with a tyrosine residue at the C-terminus, and has three Ca2+-binding sites. 

R)- and ( -selective HNLs. A number of recombinant HNLs have also been expressed in E. coli, Saccharomyces cerevisiae, and Pichia pastoris. Recently, protein engineering has been successfully applied to the development of a tailor-made HNL for large-scale production of specific cyanohydrins [69,70]. 

Lastly, it has been shown that in rare cases trisulfide bridges (Cys-SSS-Cys) can be found as hydrophobic variants of recombinant proteins. The experimental mass of such a trisulfide-bridged peptide would be increased by 32 amu. Andersson et al.125 have described this phenomenon for recombinant human growth hormone during expression in E. coli. However, the receptor binding properties were not affected by this trisulfide modification. 

Tsouloufis et al.8 used an ELISA to assess the refolding of a recombinant subunit of the extracellular domain of the human muscle acetylcholine receptor expressed in E. coli. The plates were coated with refolded or unfolded protein and then reacted with conformationally dependent MAbs. The use of specific... 

A number of B. pertussis (polypeptide) antigens have been expressed in E. coli and other recombinant systems. Several of these are being evaluated as potential subunit vaccines, including B. pertussis surface antigen, adhesion molecules and pertussis toxin. Pertussis toxin has been shown to protect mice from both aerosol and intracerebral challenge with virulent B. pertussis. The bacterial proteins that mediate surface adhesion protect mice from aerosol but not intracerebral challenge. Future pertussis subunit vaccines may well contain a combination of two or more pathogen-derived polypeptides. 

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