Proteins expression in E. coli

In the biosynthetic approach, protein expression in E. coli [27], yeast [28, 29], and plants [30, 31] have been employed. This approach requires the construction of genes encoding for these repetitive polypeptides. Different methods for gene construction have been pubhshed multimerization [32], recursive directional ligation [33], and recursive directional ligation via plasmid reconstruction [23, 34]. 

Lowe, P. A. and Rhind, S. K., Solubilization, refolding, and purification of eukaryotic proteins expressed in E. coli, in Protein Purification — Micro to Macro, Burgess, R., Ed., Alan R. Liss, New York, 1988, 429. 

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular protein production complicates downstream processing (relative to extracellular production) as ... 

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

C-terminal modification. This was caused by big problems in the synthesis and isolation of completely modified proteins [223] and good accessibility to the non-modified proteins expressed in E. coli. 

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. 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

High-Throughput Purification of Hexahistidine-Tagged Proteins Expressed in E. coli... 

Yeast display has been proposed recently as an alternative way to display mammalian proteins. Because some eukaryotic proteins expressed in E. coli are not in the soluble form, they cannot be incorporated into phage particles. Furthermore, phage display may sometimes select for reduced host toxicity or higher infectivity rather than increased affinity. Yeast display should alleviate expression biases present in E. coli and, as bacteria, have the advantage of high expression levels of displayed fusion polypeptides and selection through flow cytometric cell sorting. 

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. 

Tolbert TJ, Franke D, Wong CH. A new strategy for glycoprotein synthesis ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein. Bioorg. Med. Chem. 2005 13 909-915. 

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. 

To characterize protein-ligand interactions by F NMR, the ligand or the protein can be labeled with fluorine to produce spectra without overwhelming complexity. Proteins expressed in E.coli and tissue culture have been labeled with fluorine by biosynthetic incorporation of fluoro analogs of tryptophan, phenylalanine and tyrosine. Conformational properties of receptor... 

For a complete list of commercially available destination vectors, please visit uniw.in-vitrogen.com. These cover a broad array of applications including protein expression in E. coli, yeast, insect, and mammalian cells, yeast two hybrid protein interaction, and gene knock-down RNAi vectors. 

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