Cloning Escherichia coli cells

Zeyer, J., Lehrbach, P. R. Timmis, K. N. (1985). Use of cloned genes of Pseudomonas TOL plasmid to effect biotransformation of benzoates to cis-dihydrodiols and catechols by Escherichia coli cells. Applied and Environmental Microbiology, 50, 1409-13. 

Takahashi et al.61s further identified the primary structure by preparing a cDNA library from A. fumigatus induced with fructosylpropylamine and isolated a clone using a polyclonal Amadoriase II antibody. The structure comprised 438 amino acid residues, corresponding to 48.798 kDa. The identity of the Amadoriase n cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern-blotting analysis showed that Amadoriase II was induced by fructosylpropylamine in a dose-dependent manner. The sequence determined showed the enzyme to represent a new family of mammalian enzymes. The sequence exhibited 82 and 36% identity and 92 and 65% similarity, respectively, with the two sequences determined by Yoshida et al.616 Amadori products have been implicated in the formation of H202, but the in vivo mechanism needs to be elucidated further. 

Oguma, T., Kurokawa, T., Tobe, K., and Kobayashi, K. 1995. Cloning and sequence analysis of the cycloisomalktooloigosaccharide glucanotransferase gene from Bacillus circulans T-3040 and expression in Escherichia coli cells. Oyo Toshitsu Kagaku, 42,415-419. 

A vector used to clone DNA fragments (100- to 300-kb insert size average, 150 kb) in Escherichia coli cells. Based on naturally occurring F-factor plasmid found in the bacterium E. coli. 

A number of allergens from both honey bee and vespid venoms have been cloned and expressed by either Escherichia coli or baculovirus-infected insect cells (table 1) phospholipase Aj [20], hyaluronidase [21], acid phosphatase [13] and Api m6 [14] from honey bee venom, as well as antigen 5 [22], phospholipase A and hyaluronidase [23] from vespid venom, and dipeptidylpeptidases from both bee and Vespula venoms [15, 16]. Their reactivity with human-specific IgE antibodies to the respective allergens has been documented [11-16, 22, 23] and their specificity is superior... 

Oldfield s work confirmed the complete pathway and identified all the intermediates via an exhaustive experimental scheme, which included whole cell assays, cell-free extracts of IGTS8 as well as extracts from clones containing individual genes expressed in Escherichia coli. The need for NADH was clearly demonstrated in cell-free assays by amendment of NADH. The experimental evidence for involvement of FMN in the pathway was demonstrated [53,66,67],... 

The enzyme catalyzing the formation of retinal 2 by means of central cleavage of P-carotene 1 has been known to exist in many tissues for quite some time. Only recently, however, the active protein was identified in chicken intestinal mucosa (3) following an improvement of a novel isolation and purification protocol and was cloned in Escherichia coli and BHK cells (4,5). Iron was identified as the only metal ion associated with the (overexpressed) protein in a 1 1 stoichiometry and since a chromophore is absent in the protein heme coordination and/or iron complexation by tyrosine can be excluded. The structure of the catalytic center remains to be elucidated by X-ray crystallography but from the information available it was predicted that the active site contains a mononuclear iron complex presumably consisting of histidine residues. This suggestion has been confirmed by... 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [>600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

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