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PubMed2002

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently.

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

Amino Acid Sequence↗
PubMed2006

RAISE: a simple and novel method of generating random insertion and deletion mutations.

Although proteins may be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult, and the mutations introduced are no more variable than those introduced by the introduction of random point mutations. We describe here a three-step method called RAISE, which is based on gene shuffling and can introduce a wide variety of insertions, deletions and substitutions. To test the efficacy of this method, we used it to mutate TEM beta-lactamase to generate improved antibiotic resistance. Some unique insertion or deletion mutations were observed in the improved mutants, some of which caused higher activities than point mutations. Our findings indicate that the RAISE method can yield unique mutants and may be a powerful technique of protein engineering.

Amino Acid Substitution↗
PubMed1993

Retroviral DNA integration: lessons for transposon shuffling.

Phylogenetic comparisons of retroviral IN (integrase) protein sequences have revealed homologies that extend to the retrotransposon and bacterial transposase families and have provided evidence for at least two functional domains. The N-terminal region is characterized by a Zn-finger-like array which is conserved in retrotransposons. The central region is defined by a D,D(35)E amino acid constellation which is conserved through the retrotransposons and several bacterial IS element transposases. Mutagenesis studies and biochemical analysis of the isolated central D,D(35)E domain support our original suggestion that this region contains the catalytic center of these proteins which must all share a similar enzymatic mechanism. These, and other recent findings suggest unexpected relationships between diverse pathways of nucleic acid metabolism in eukaryotes and prokaryotes.

DNA Nucleotidyltransferases↗
PubMed2005

Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains.

We have developed a class of binding proteins, called avimers, to overcome the limitations of antibodies and other immunoglobulin-based therapeutic proteins. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains creates avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages over immunoglobulin domains include simple and efficient production of multitarget-specific molecules in Escherichia coli, improved thermostability and resistance to proteases. Avimers with sub-nM affinities were obtained against five targets. An avimer that inhibits interleukin 6 with 0.8 pM IC50 in cell-based assays is biologically active in two animal models.

Animals↗
PubMed2005

Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli.

Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05-0.7% based on the number of protoplasts subjected to fusion. This frequency is three-four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens.

Artificial Gene Fusion↗
PubMed2003

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity.

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Amino Acid Sequence↗
PubMed2006

Shuffling of discrete tRNASer regions reveals differently utilized identity elements in yeast and methanogenic archaea.

Seryl-tRNA synthetases (SerRSs) from methanogenic archaea possess distinct evolutionary origin and show minimal sequence similarity with counterparts from bacteria, eukaryotes and other archaea. Here we show that SerRS from yeast Saccharomyces cerevisiae and archaeon Methanococcus maripaludis (ScSerRS and MmSerRS, respectively) display significantly different ability to serylate heterologous tRNA(Ser). Recognition in yeast was shown to be more stringent than in archaeon. While cross-aminoacylation of M. maripaludis tRNA(Ser) (MmtRNA(Ser)) by yeast SerRS barely occurs, yeast tRNA(Ser) (SctRNA(Ser)) was shown to be a good substrate for heterologous MmSerRS. To investigate the contribution of different tRNA regions for the recognition by yeast and archaeal SerRS, chimeric tRNAs bearing separated domains of SctRNA(Ser) in MmtRNA(Ser) framework were produced by in vitro transcription and subjected to kinetic and gel mobility shift analysis with both enzymes. Generally, the recognition in M. maripaludis seems to be relatively relaxed toward tertiary elements of tRNA(Ser) structure and relies on the direct recognition of identity nucleotides. On the other hand, expression of tRNA(Ser) identity elements in yeast seems to be more sensitive toward surrounding sequence context. In both systems variable arm of tRNA was recognized as a major identity region with a strong influence on SerRS:tRNA binding. Acceptor domain of SctRNA(Ser) was also shown to be important for serylation in yeast. We propose that cognate interactions between N-terminal domain of yeast SerRS and variable region of SctRNA(Ser) place the acceptor stem into the enzyme's active site and lead to increased affinity toward serine and efficient serylation of tRNA. The same effect was not observed in M. maripaludis. Unlike its yeast counterpart, MmSerRS forms only one type of covalent complex with MmtRNA(Ser), regardless of the tRNA/SerRS molar ratio. Stoichiometry of the complex, one tRNA per dimeric SerRS, was revealed by mass spectrometry. Our studies indicate that different SerRS:tRNA recognition mode is utilized by these two systems.

Base Sequence↗
PubMed2004

Addressing the challenge of changing the specificity of RNase T1 with rational and evolutionary approaches.

Although ribonuclease T1 (RNase T1) is one of the best-characterized proteins with respect to structure and enzymatic action, numerous attempts at altering the specificity of the enzyme to cleave single-stranded RNA at the 3'-side of adenylic instead of guanylic residues by rational approaches have failed so far. Recently we generated and characterized the RNase T1 variant RV with a 7200-fold increase in adenylyl-3',5'-cytidine (ApC)/guanylyl-3',5'-cytidine (GpC) preference, with the guanine-binding loop changed from 41-KYNNYE-46 (wt) to 41-EFRNWN-46. Now we have introduced the asparagine residue at position 46 of the wild-type enzyme as a single-point mutation in variant E46N and in combination with the Y45W exchange also occurring in RV. Both variants show an improved ApC/GpC preference with a 1450-fold increase for E46N and a 2100-fold increase for Y45W/E46N in comparison to wild-type activity. We also addressed the challenge of altering enzyme specificity with an evolutionary approach. We have randomly introduced point mutations into the RNase T1 wild-type gene and into the gene of the variant RV with different mutation rates. Altogether we have screened about 100,000 individual clones for activity on RNase indicator plates; 533 of these clones were active. A significant change in substrate specificity towards an ApC preference could not be observed for any of these active variants; this demonstrated the magnitude of the challenge to alter the specificity of this evolutionary perfected enzyme.

Amino Acids↗
PubMed2004

Domain exchange between isoforms of ferredoxin-NADP+ reductase produces a functional enzyme.

Two isoforms of ferredoxin-NADP(+) reductase (FNR) exist in higher plants, the leaf (or photosynthetic) and the root (or non-photosynthetic) isoform, which have 48% amino acid sequence identity and display specific structural and functional features. With the aim to gain further insight into the structure-function relationship of this enzyme, we designed two novel chimeric flavoenzymes by swapping the structural domains between the leaf and the root isoforms. Characterization of the chimeras would allow dissection of the contribution of the individual domains to catalysis. The chimera obtained by grafting together the FAD-binding domain of the root-isoform and the NADP-binding domain of the leaf-isoform was inactive when expressed in Escherichia coli. On the other hand, the chimera assembled in the opposite way (leaf FAD-binding domain and root NADP-binding domain) was functional and was produced in the bacterial host to a level threefold higher than that of the parent enzymes. The protein was purified and found to be as stable as the natural isoforms. Limited proteolysis excluded the presence in the chimera of misfolded regions. The affinity of the chimera for ferredoxin I (Fd I) was similar to that of the leaf isoform, although interprotein electron-transfer was partially impaired. As occurs with the root isoform, the chimera bound NADP(+) with high affinity, while spectroscopic evidence suggested that the conformation adopted by the nicotinamide moiety bound to the chimera was similar to that observed in the leaf enzyme. Interestingly, the chimera, by combining favorable features from both parent isoforms, acquired a catalytic efficiency (k(cat)/K(m)), as an NADPH-dependent diaphorase, higher than those of both the root ( approximately 2-fold) and the leaf enzyme ( approximately 5-fold). Thus, molecular breeding between isozymes has improved the catalytic properties of FNR.

Catalysis↗
PubMed2005

Mining genomes and 'metagenomes' for novel catalysts.

Advances in the field of genomics and 'metagenomics' have dramatically revised our view of microbial biodiversity and its potential for biotechnological applications. Considering the estimation that >99% of microorganisms in most environments are not amenable to culturing, very little is known about their genomes, genes and encoded enzymatic activities. The isolation, archiving and analysis of environmental DNA (or so-called 'metagenomes') has enabled us to mine microbial diversity, allowing us to access their genomes, identify protein coding sequences and even to reconstruct biochemical pathways, providing insights into the properties and functions of these organisms. The generation and analysis of (meta)genomic libraries is thus a powerful approach to harvest and archive environmental genetic resources. It will enable us to identify which organisms are present, what they do, and how their genetic information can be beneficial to mankind.

Biotechnology↗
PubMed2021

Novel human liver-tropic AAV variants define transferable domains that markedly enhance the human tropism of AAV7 and AAV8.

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. Here, we report the bioengineering of a set of next-generation AAV vectors, named AAV-SYDs (where "SYD" stands for Sydney, Australia), with increased human hepato-tropism in a liver xenograft mouse model repopulated with primary human hepatocytes. We followed a two-step process that staggered directed evolution and domain-swapping approaches. Using DNA-family shuffling, we first mapped key AAV capsid regions responsible for efficient human hepatocyte transduction in vivo. Focusing on these regions, we next applied domain-swapping strategies to identify and study key capsid residues that enhance primary human hepatocyte uptake and transgene expression. Our findings underscore the potential of AAV-SYDs as liver gene therapy vectors and provide insights into the mechanism responsible for their enhanced transduction profile.

AAV↗
PubMed2005

Methods for the study of alternative splicing.

Alternative splicing multiplies the coding capacity of genes, which provides unparalleled complexity to the transcriptome and proteome. This increased complexity has clear repercussions for the regulation of gene expression in many organisms and for the balance between human health and disease. This significance, which has only recently been fully understood, has led to a growing number of studies that address the mechanisms that control alternative splicing decisions and the biological consequences of alternative RNA and protein isoforms. These studies have adapted or developed new methods and protocols that span computational biology, the biochemical analysis of in vitro reactions, model tissue culture systems, and in vivo studies. Many of these methods have been assembled in this volume to assist a newcomer to the study of alternative splicing and to expand the methodologies of laboratories already in the field.

Alternative Splicing↗
PubMed2003

Transgenic animals in biomedicine and agriculture: outlook for the future.

Transgenic animals are produced by introduction of 'foreign' deoxyribonucleic acid (DNA) into preimplantation embryos. The foreign DNA is inserted into the genetic material and may be expressed in tissues of the resulting individual. This technique is of great importance to many aspects of biomedical science including gene regulation, the immune system, cancer research, developmental biology, biomedicine, manufacturing and agriculture. The production of transgenic animals is one of a number of new and developing technologies that will have a profound impact on the genetic improvement of livestock. The rate at which these technologies are incorporated into production schemes will determine the speed at which we will be able to achieve our goal of more efficiently producing livestock, which meets consumer and market demand.

Agriculture↗
PubMed2003

From plant genomics to breeding practice.

New alleles are constantly accumulated during intentional crop selection. The molecular understanding of these alleles has stimulated new genomic approaches to mapping quantitative trait loci (QTL) and haplotype multiplicity of the genes concerned. A limited number of quantitative trait nucleotides responsible for QTL variation have been described, but an acceleration in their rate of discovery is expected with the adoption of linkage disequilibrium and candidate gene strategies for QTL fine mapping and cloning. Additional layers of regulatory variation have been studied that could also contribute to the molecular basis of quantitative genetics of crop traits. Despite this progress, the role of marker-assisted selection in plant breeding will ultimately depend on the genetic model underlying quantitative variation.

Breeding↗
PubMed2003

Novel carotenoids genetically engineered in a heterologous host.

Carotenoids are commercially important pigments that are essential for human health. Diverse carotenoids have been identified, but availability has impeded evaluation of their pharmaceutical potential. Molecular techniques have been developed to produce specific and novel carotenoids with improved biological activities.

Carotenoids↗
PubMed2003

Substrate turnover and inhibitor binding as selection parameters in directed evolution of blood coagulation factor Xa.

A library of blood coagulation factor Xa (FXa)-trypsin hybrid proteases was generated and displayed on phage for selection of derivatives with the domain "architecture" of trypsin and the specificity of FXa. Selection based on binding to soybean trypsin inhibitor only provided enzymatically inactive derivatives, due to a specific mutation of serine 195 of the catalytic triad to a glycine, revealing a significant selection pressure for proteolytic inactive derivatives. By including a FXa peptide substrate in the selection mixture, the majority of the clones had retained serine at position 195 and were enzymatically active after selection. Further, with the inclusion of bovine pancreatic trypsin inhibitor, in addition to the peptide substrate, the selected clones also retained FXa specificity after selection. This demonstrates that affinity selection combined with appropriate deselection provides a simple strategy for selection of enzyme derivatives that catalyse a specific reaction.

Amino Acid Sequence↗