Codon Devices described Constructive Biology™ as an enabling technology for engineering biological products such as genes, proteins, metabolic pathways, and cell lines. The company’s BioFAB™ DNA-assembly platform produces libraries of defined DNA sequences in lengths from 1,000 to upwards of 35,000 base pairs.
Dasa Lipovsek, Ph.D., director of protein engineering, described the company’s new BioLOGIC™ protein engineering platform, which is based on the ability of BioFAB to design and construct synthetic DNA genes accurately and inexpensively.
“We can make a large DNA library with defined sequences that accurately reflect the design rules,” said Dr. Lipovsek. Utilizing computational, modeling, and analytical tools, BioFAB generates sets of thousands to a hundred thousand predictive sequences.
In contrast to approaches based on random mutagenesis, Codon Devices employs a directed library design method to produce custom-defined sequences that incorporate mutations at specified rates and positions, she added. DNA error filtration using immobilized MutS to detect mismatched base pairs leads to removal of the faulty double stranded DNA sequence from further assembly.
BioFAB is at the core of the BioLOGIC process, which also relies on structure-based computational protein design tools to generate 3-D protein models capable of depicting molecular interactions at an atomic level. Algorithms then predict the amino acid changes intended to result in a function of interest.
Once a library of predictive genes is produced and cloned into a host, the company uses a cell-surface display platform for high-throughput selection of the proteins of interest. Each protein remains associated with its corresponding DNA sequence, so when we “select a protein, the gene comes along for the ride,” said Dr. Lipovsek.
She described how the company’s secretion-and-capture selection method enables the yeast cell to secrete the protein, which adheres, by means of a sticky label, to the outside of the cell, with the plasmid/oligonucleotide remaining inside the cell. By exposing the protein-studded yeast to a target antigen one can characterize the proteins for binding affinity, activity, and stability, select the proteins of interest, and extract and sequence the DNA. Next, the selected gene is used to express sufficient protein for further characterization.
“We then put the oligo into a new host, express the protein,” and produce enough for biochemical characterization studies, she explained.
In addition to ongoing internal projects, Codon Devices is involved in collaborative efforts focusing on therapeutic protein discovery and lead optimization as well as biofuels development.
Designed for high-throughput screening to identify the optimal conditions for refolding of a particular protein, EMD Novagen iFOLD™ systems allow researchers to perform protein refolding screens in a 96-well format. Achieving high yield of recombinant proteins in E. coli-based expression systems is relatively straightforward and economical, but producing properly folded proteins required for functional and structural studies is often challenging, according to Alla Zilberman, Ph.D., director of product management at Novagen.
A substantial amount of the desired protein output may be present in inclusion bodies, which are dense, insoluble aggregates of misfolded proteins. These can be solubilized and purified, “but defining conditions that promote refolding of a chemically denatured protein into its native conformation is empirical and often time-consuming,” noted Dr. Zilberman. iFOLD technology allows for systematic evaluation in parallel of a large number of refolding conditions, she added.
EMD launched its iFOLD System 3, which assays 96 different refolding conditions and incorporates Triton-X detergent to wash the inclusion bodies and novel FoldACE™ additives that enhance protein refolding and stabilize the refolded proteins. The company’s iFOLD System 2 product is a detergent-free screening system.
At the meeting, Sidec elaborated on protein tomography, which provides 3-D visualization of individual antibody molecules bound to a target, according to Michael Smith, Ph.D., field scientist. The key feature of the technology, Dr. Smith explained, is single molecule detection and visualization, which enables both epitope mapping and mechanism-of-action studies. The research is able to determine antibody-binding sites by direct inspection of the binding event in three dimensions, and to detect conformational changes of individual target molecules in the presence of an agonist or antagonist, allowing the study of how binding affects target function. With a resolution of 25-30 Å, protein tomography can be used with purified protein and protein complexes or to view where and how tagged antibodies bind to their targets inside cells.
“We work with our clients to develop a better IP strategy and an improved product positioning for therapeutic antibodies through improved drug candidate selection, lead classification, and target mechanism studies,” said Dr. Smith.