Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.
DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination, combining useful mutations from individual genes. Libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. Template switching causes crossovers in areas of sequence homology. Our previous studies used single genes and random point mutations as the source of diversity. An alternative source of diversity is naturally occurring homologous genes, which provide 'functional diversity'. To evaluate whether natural diversity could accelerate the evolution process, we compared the efficiency of obtaining moxalactamase activity from four cephalosporinase genes evolved separately with that from a mixed pool of the four genes. A single cycle of shuffling yielded eightfold improvements from the four separately evolved genes, versus a 270- to 540-fold improvement from the four genes shuffled together, a 50-fold increase per cycle of shuffling. The best clone contained eight segments from three of the four genes as well as 33 amino-acid point mutations. Molecular breeding by shuffling can efficiently mix sequences from different species, unlike traditional breeding techniques. The power of family shuffling may arise from sparse sampling of a larger portion of sequence space.
Transferred DNA (T‐DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T‐DNA‐tagged genomic loci showed that T‐DNA integration resulted in target site deletions of 29–73 bp. In those cases where integrated T‐DNA segments turned out to be smaller than canonical ones, the break‐points of target deletions and T‐DNA insertions overlapped and consisted of 5–7 identical nucleotides. Formation of precise junctions at the right T‐DNA border, and DNA sequence homology between the left termini of T‐DNA segments and break‐points of target deletions were observed in those cases where full‐length canonical T‐DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T‐DNA segments showed no homology to break‐points of target sequence deletions. Homology between short segments within target sites and T‐DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T‐DNA integration results from illegitimate recombination. The data suggest that while the left T‐DNA terminus and both target termini participate in partial pairing and DNA repair, the right T‐DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.
A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.
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