Another extensively utilized yeast expression system is that of Saccharomyces cerevisiae. It has become a platform organism in the field of metabolic engineering, which seeks to improve cellular activities and properties (e.g., the production of a metabolite) by genetic manipulation.
Nancy DaSilva, Ph.D., professor, chemical engineering and materials science, biomedical engineering, and Suzanne Sandmeyer, Ph.D., professor, biological chemistry, University of California, Irvine have developed a vector toolkit for systematic pathway engineering in S. cerevisiae.
Although scientists often study the introduction of individual genes using vectors and PCR-based integration, metabolic engineering typically requires the regulated expression of multiple genes to better optimize both pathway expression and function. To avoid instability, the integration of multiple expression cassettes may be required.
Drs. DaSilva and Sandmeyer and colleagues constructed a set of expression vectors for metabolic engineering applications. To promote differential expression of genes, six different promoters and six different reusable selection markers were included. The vectors were designed to allow the seamless transition from plasmid-based expression to PCR-based chromosomal gene integration.
Expression from the vectors and from multiple different integration sites has been characterized. According to Dr. DaSilva, the methodology can facilitate rapid and systematic combinatorial expression of pathway genes.
Deriving monoclonal antibodies (mAb) for therapeutic development can be like finding a needle in a haystack. Masaharu Isobe, Ph.D., professor of molecular and cellular biology, life sciences and bioengineering, University of Toyama, has developed a semi-automatic way for high-throughput generation of mAbs.
Quick mAb Production
“Although single-cell immunoglobulin variable gene cloning is the best way to generate recombinant monoclonal antibodies, these methods remain an obstacle to the rapid and high-throughput production of mAbs, particularly because of difficulties in stable amplification of the V genes from single cells and tedious cloning steps to obtain proper immunoglobulin gene-expression constructs.
“We developed a novel overlap extension PCR methodology in conjunction with a new instrument for 5´RACE-ready cDNA synthesis that produces a large number of recombinant mAbs very quickly.”
Dr. Isobe’s group developed a robotic magnetic bead-handling instrument. “Single cell-based cDNA synthesis is a required fine art due to the limited amount of source. To automatize cDNA synthesis from large numbers of single cells, we constructed a noncontact magnetic power transmission instrument that we call the MAGrahder.
“It has MAGrahder reactor trays and a desktop robot. It also has 12-channel, parallel magnetic rods on a robotic arm that transports and mixes nucleic acid-bound magnetic beads in the trays. This is followed by mRNA extraction, reverse transcription, and the homopolymer-trailing reaction that can handle up to 144 samples and only takes an hour to complete.”
However, according to Dr. Isobe, a second challenge was the need for a simple and efficient way to make expression constructs. Dr. Isobe’s method, called target selective joint polymerase chain reaction (TS-jPCR), amplifies gene fragments and assembles them into an immunoglobulin-expression construct without any purification steps.
“We join the 3´-random nucleotide-tailed V gene fragments produced by PCR with a specific immunoglobulin cassette containing all the necessary elements for antibody expression. The resulting immunoglobulin-expression constructs from amplified V genes are ready to transfect into cultured cells without any purification, and the media is used for the analysis of binding specificities of mAbs,” says Dr. Isobe.
“Our new system significantly reduces the cost and the time for generation of mAbs and allows us to obtain hundreds of mAbs within four to five days.”