The existing workflow must be redesigned to a cost-effective, scalable process in which, the untagged protein is recombinantly expressed in E. coli and undergoes in vivo maturation. The first step in achieving this is the optimization of expression parameters to enrich the mature form of the protein in the soluble cytoplasmic fraction. Following this, the scalable purification strategy designed, needs to be addressing the quality and quantity specifications of the final protein. This case study demonstrates the experience and expertise of scientists at Aragen that helped the client in re-shaping their strategy to reach a critical milestone.
The client was looking at obtaining >50 mg of a mammalian secretory protein which is known to be difficult to express as a soluble form in E. coli. Aragen was approached to recombinantly produce this protein in its precursor form in E. coli, facilitate in vivo maturation by co-expression of a processing enzyme, and produce the tag-less protein with >95% purity. This project was a crucial step for the client to initiate a new program in their drug discovery pipeline.
The client is a biotechnology company focussed on protein engineering to create multifunctional, precision therapies for various indications
The initial trial protocol provided by the client was further modified and optimized to achieve required quality and quantity of the final product. As a first step, the scientific team at Aragen re-worked the expression protocol to maximize the yield of the soluble protein. This was followed by redesigning a purification protocol to address the specifications in a scalable format with reduced turn-around time
The expression and purification protocol were redesigned at Aragen to address the target quantity and purity of the deliverable. In a series of small – scale experiments (<1 L cultures), optimal temperature for expression was determined which enhanced maximal soluble expression of the precursor protein and provided optimal in vivo reaction condition for the co-expressed processing enzyme to convert the soluble precursor protein into its mature form. A 4-step purification methodology was developed to obtain the final product with the desired purity in the shortest timeframe. Optimized parameters were tested for scalability (up to 10 L per batch) and reproducibility. More than 80 mg of the protein, meeting desired specifications, was produced, and delivered to the client.
Step 1: Expression (<1 L scale)
Step 2: Purification
Step 3: Characterization and QC
Step 4: Large – scale expression and purification (>10 L scale)
A robust, scalable, and reproducible process was designed for expression and purification of a recombinant protein in bacterial (E. coli) system. Expression parameters were thoroughly optimized to enrich the mature form of the protein in the soluble cytoplasmic fraction. A scalable purification strategy was designed to addresses the quality and quantity specifications of the final recombinant protein. More than 80 mg of the protein, meeting desired specifications, was produced, and delivered to the client.
