Recombinant Mammalian Proteins
Ashok Bandaranayake, Ph.D., associate in the department of biochemistry at the Albert Einstein College of Medicine, will discuss Daedalus, which was developed for rapid production of recombinant mammalian proteins in human cell culture. Dr. Bandaranayake started this work when he was at the Seattle Children’s Hospital Research Institute.
He is currently optimizing Daedalus for a high-throughput protein production effort as part of the Protein Structure Initiative (New York Structural Genomics Consortium). He employs the Lentivirus system, which can be used to efficiently transduce human cells.
This low copy number vector (10–20 copies per genome) also has package-size limitations. With clones greater than 10 kb the viral titer drops significantly, and it has been observed that efforts to increase the copy number often result in a net negative result with increased promoter silence when incorporated into the genome.
Dr. Bandaranayake has overcome the low copy number by using a strong promoter (from spleen focus forming virus). He has overcome promoter silence observation by adding a minimized ubiquitous chromatin opening element. These GC-rich sequences exist as islands of 8 kb, 4 kb, or 2 kb, all of which impact the package-size limitations.
Dr. Bandaranayake has also cloned a 0.7 kb element that is equally effective in keeping expression levels high. To effectively overcome the package-size limitation, Dr. Bandaranayake has demonstrated that cells can be co-infected with vectors containing different subunits of a large protein.
These subunits have been shown to readily self-assemble in the positively transfected cells. The final trick is the cloning of signal peptide encoding sequences upstream of the gene of interest to make sure the produced recombinant protein is secreted into the media.
Protein secretion ensures that the protein is folded correctly and makes for easy purification. Proteins purified from serum-free media come off as a single peak from gel filtration columns. “We use serum-free adapted HEK293 cells for production of the recombinant proteins,” Dr. Bandaranayake notes.
“We have demonstrated the utility of this system using soluble immune receptors, six different human cytokines, and most recently two canine cytokines that are used in transplant studies. This approach is not applicable for membrane proteins as the essential element is the secretion of the protein product. You would have to mutate the membrane-binding domains to make sure the integral membrane protein didn’t get hung up in the membrane. By the time you’ve done that you’ve destroyed the structure/function paradigm.”