Many biotechnological applications are based on the availability of functionally altered proteins that differ from nature’s prototypes in their improved specific activity, their better stability, enhanced affinity and other essential functions. Industrial applications for such molecules range from, for example, serving as catalysts in chemical synthesis, food fermentation and bio fuel engineering.
Therapeutic proteins are used to help patients suffering from cancer, cystic fibrosis, diabetes, Rheumatoid Arthritis and several other diseases.
Directed molecular evolution has become a very powerful strategy for changing or optimising the properties of proteins. Typically, a gene encoding the protein of interest is mutated to generate a library of mutant genes. After expression of all mutant genes in a suitable host system the resulting proteins are screened for the desired properties.
Various screening system, including phage display, ribosomal display, and in vitro compartmentalisation, have been shown to satisfy the essential requirement for a meaningful linkage of genotype and phenotype.
Subsequently, genes for selected proteins are amplified, and brought to further rounds of mutagenesis, expression and selection (Fig1). Numerous of such protein engineering experiments have demonstrated that, very often, cumulative effects of several mutations in a gene sequence may finally result in a significant change in the enzymatic functions. However, for efficient evolution experiments, it is important that the genetic diversification step yields in high-quality gene mutant libraries containing individual molecules that, with the utmost probability, display the desired functional properties. In general, methods for creating gene mutant libraries can be divided in three categories:
- Random substitution methods, such as the various protocols of error-prone PCR (ePCR), introduce changes at positions throughout the entire DNA sequence during several rounds of gene amplification.
- Recombination techniques, such as DNA shuffling approaches, take portions of existing gene variants or homologous genes, and mix them in novel combinations.
- Directed methods involve the incorporation of partially randomised synthetic DNA cassettes into genes via PCR or direct cloning. Here, genetic diversity can be generated at specific positions within the synthetic cassettes during the chemical synthesis of ‘wobble’ oligo nucleotides.
All these methods bear significant constraints regarding the possibility to sufficiently control the number and quality of mutations. Due to several sub-optimal factors, eg the characteristics of the polymerases used for gene amplification, the variable quality of chemical oligo nucleotides, or the degenerated nature of the genetic code, the resulting mutant libraries may contain only a fraction of the desired mutations, possess unfavourable changes, or yield in an uneven representation of mutants at the protein level. In consequence, the following screening process turns out to be more elaborate, time-consuming and ineffective than necessary.
Based on its patented Slonomics gene synthesis technology, Sloning BioTechnology is pursuing a novel strategy for tailoring genetic diversity. In cases where sufficient information about the structural-functional relationship is available, SlonoMax synthetic mutant libraries provide a much more promising approach for combining rational protein design strategies with the possibility to create gene variants in parallel during a highly controlled production process.
Slonomics provides an enabling platform technology for strategic protein design. The method is based on the use of double-stranded DNA triplets that can be assembled to almost any desired gene construct in a unique enzymatic protocol.
Using defined sets of such universal DNA building blocks, multiple codons can be introduced in parallel during the synthesis of a gene construct at any desired sequence position. This results in the parallel production of gene mutant variants that can be combined in a SlonoMax mutant library.
Due to the absence of any functional bias during the synthesis process, and the freedom to select up to 20 specific codons per sequence position in almost any desired proportion, the quality of the resulting mutant composition is already clearly defined by the initial work flow for production, and will also be fully maintained on protein level.
The method provides full control over the production process yielding in SlonoMax mutant libraries that correspond precisely to the previously developed theoretical design. Missing or undesired mutations are no longer limiting the following screening process. Also, the level of genetic diversity that can be obtained in SlonoMax libraries (up to approximately 1011 molecules per library) is sufficient for even utilising high-throughput screening systems.
The design potential of SlonoMax libraries varies from single site scanning strategies, the individual randomisation of selected sequence positions or the complete randomisation of stretches comprising more than eight consecutive sequence positions (Fig.2). This clearly exceeds the possibilities and quality levels that, as of today, can be reached with oligonucleotide-based production methods.
Since for the production of synthetic SlonoMax mutant libraries no already existing gene construct is required, even new genetic designs, eg constructs that are optimised for expression in a desired host systems, may easily serve as starting point for improved protein optimisation strategies.
Dr Thomas Waldmann is with Sloning BioTechnology, Munich, Germany.
www.sloning.com
