The resulting freedom to completely change the product’s amino acid sequence allows Amunix to optimize products for all desired properties such as stability, expression, formulation; whatever the therapeutic area calls for. “The product format is also designed to support optimal patent strategies,” Dr. Schellenberger concluded.
Novel E. coli Expression System
“You look at the number of microbial expression systems out there and wonder why there are so many of them,” Miguez pointed out. “The short answer to that is that, while an expression platform for protein x may work beautifully, when you move to protein y, it doesn’t work quite as well.”
Miguez and his group developed a gene-expression system for E. coli that could replace the IPTG-based platforms currently available. “The new system is capable of higher product yields compared to IPTG and uses p-cumate, a nontoxic, easy-to-handle, and cost-competitive inducer that can be used with popular strains of E. coli,” Miguez explained. “Applications include production of biopharmaceutical proteins, production of industrial enzymes, and basic and applied use in universities and research institutes.”
This E. coli gene-expression system is based on the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using cumate as an inducer. It includes a specific expression vector, pNEW, that contains a partial T5 phage promoter fused to a repressor binding DNA fragment (operator) and the repressor gene cymR driven weakly by a kanamycin promoter PKm designed to express the repressor gene constitutively in the E. coli host strain.
“The exogenous inducer p-cumate is necessary for the induction of transcription,” Miguez explained. “The very tightly cumate-regulated expression system can be used with popular E. coli strains and potentially produce any gene that can be expressed using IPTG-inducible systems.”
Miguez noted that his E. coli gene-expression system is capable of high induction of transcription and low basal expression, surpassing the yields of IPTG-based gene expression systems. “The expression vector can be used with popular strains such as BL-21 (DE3), enabling those currently using IPTG-based systems to adopt the novel E. coli gene-expression system.”
One key feature Miguez commented on was that this expression vector is a viable alternative to ITPG-based systems that do not express some genes well, leading to insoluble or incorrectly folded proteins. Miguez’ E. coli gene-expression system may be an avenue for the production of these types of genes. “There is a lot of interest, and several companies want to try it out. Expression is gene dependent, which is why there are so many expression systems.”
Optimization in Baculoviral Expression System
Jim King, Ph.D., principal scientist for Boehringer-Ingleheim, and his group use the baculoviral expression system for optimization of proteins for crystallography. He calls this expression system the workhorse platform in both industry and academia for the last 15 years. “Until about five years ago,” Dr. King added, “it was really just a production system. In the last five years, it’s really advanced to screening a much larger number of constructs.”