Contribution

Wet Lab Protocols

Protein purification pipeline

1. Cloning

   Purifying a protein of interest from bacteria first requires designing, assembling, and transforming a genetic construct that codes for the desired protein and contains conditions for when the cell is to express that protein. For our project, we aimed to express three different transaminase enzymes, and so designed and assembled three separate genetic constructs - one to produce each enzyme. We designed our genetic construct to be an IPTG inducible system and tagged our protein with histidine to aid in purification of the protein. More information about the design of our part can be found below, or on the iGEM registry. In the first phase of protein purification, cloning, you will grow bacteria containing the expression vector backbone, then extract the backbone from the culture. You will then digest the backbone using restriction enzymes to create homology regions with the gene block insert coding for the protein of interest. After confirming proper digestion of the backbone, you will purify the linearized backbone and assemble it with the insert to create your expression vector. You will then create competent cells in which to mass-produce your assembled expression vector and to grow the protein in for extraction. After completing all of this, you will be ready to move on to expressing and purifying your protein of interest!

2. Protein expression and purification

   Following the successful assembly and transformation of the genetic construct in Phase I, the project proceeds to Phase II: Protein Expression and Purification. This stage is pivotal in translating the genetic information into a functional protein, which in the context of our iGEM project, are transaminase enzymes used for levothyroxine synthesis. In this phase, the previously transformed protein expression strain, for our project specifically the BL21 strain of E. coli, becomes the focal point. It serves as the cellular factory for producing the protein of interest. Initiating this phase is the introduction of the assembled expression vector into the chosen expression strain. Before scaling up, it is advisable to conduct a test expression to verify both the induction and the expression of the intended protein. We employed an IPTG-inducible system for this purpose, and the protocols are tailored accordingly. After confirming successful test expression, the project moves on to large-scale protein expression and purification. Here, the histidine tag incorporated into our genetic construct plays a crucial role in aiding protein purification, which often involves techniques like affinity chromatography. By diligently executing this phase, you effectively bridge the gap between the theoretical genetic construct and its tangible protein product. This forms the basis for the subsequent Phase III, which involves the rigorous characterization of the produced protein to assess its enzymatic activity and functional integrity.

3. Protein characterization

   Upon successful expression and purification of the target protein, the next pivotal phase is its characterization. This is a critical step as it validates the functional integrity of the protein and provides insights into its enzymatic activity. In the case of our project, this is particularly relevant because the transaminase enzymes are the linchpin of our synthetic pathway for levothyroxine synthesis. The characterization phase aims to assess the protein's enzymatic activity, validate its functional domains, and ensure that the purified protein meets the desired criteria for subsequent application. In our project, we utilized a UV-vis assay to measure the enzymatic activity of transaminases. The characterization phase involves a series of intricate procedures designed to elucidate the protein's functionality and suitability for its intended role. For the transaminase enzymes, we have employed a UV-vis assay to evaluate enzymatic activity. This assay is essential for confirming that the transaminases are active and capable of catalyzing the transamination reactions necessary for levothyroxine synthesis. UV-vis and other protein characterization methods not only confirm the success of the preceding cloning and purification phases but also provides the foundational understanding of the enzyme’s functionality, a critical factor in the overall success of our synthetic pathway for levothyroxine production and for most iGEM projects involving the catalytic activity of proteins.