Disease relevance of DNA Shuffling

• Using the process of DNA shuffling, we randomly recombined the bphA1 genes of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400 and selected for genes that expressed proteins with altered function [1].
• We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity [2].
• This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display [3].
• The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling [4].

High impact information on DNA Shuffling

• The technique of plasmid-assisted molecular breeding has led to the development of bacterial strains capable of totally degrading 2,4,5-T by using it as their sole source of carbon at high concentrations (greater than 1 mg/ml) [5].
• Molecular breeding of carotenoid biosynthetic pathways [6].
• To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling [7].
• Molecular evolution of an arsenate detoxification pathway by DNA shuffling [8].
• After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k(cat)/K(m) toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity [9].

Biological context of DNA Shuffling

• The bglB gene from Paenibacillus polymyxa was subjected to random mutagenesis mediated by error prone polymerase chain reaction amplification and DNA shuffling [10].
• Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations [11].
• Functional evolution of the substrate specificity of biphenyl dioxygenase was demonstrated by DNA shuffling and staggered extension process (StEP) of the bphA gene coding for the large subunit of biphenyl dioxygenase [12].
• Random DNA fragmentation with endonuclease V: application to DNA shuffling [13].

Associations of DNA Shuffling with chemical compounds

• Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round [14].
• Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro [15].
• One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides [16].
• After three rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate [17].
• A derivative of the TEM-1 beta-lactamase producing clinically significant levels of resistance to ceftazidime and beta-lactamase inhibitors in the presence of penicillins was generated following five rounds of DNA shuffling and selection [18].

Gene context of DNA Shuffling

• We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening [19].
• To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used [20].
• DNA shuffling of mutations from the various screens and recombination onto a T169E-encoding 3' end yielded CDC28 mutants with strong T169E suppression [21].
• To understand the molecular basis by which diarylpropionitrile (DPN), an ERbeta-selective ligand, is able to discriminate between the two ERs, we examined its activity on ER mutants and chimeric constructs generated by DNA shuffling [22].
• DNA shuffling method for generating estrogen receptor alpha and beta chimeras in yeast [23].

Analytical, diagnostic and therapeutic context of DNA Shuffling

• Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling [4].

References