Please refer to our protocol section for detailed steps of our experiments.

Experimental Design

Our experiment consists of three main sections, synthesizing the modified protein, induce the binding of different components, and testing the affinity of our ABM design.

Protein Synthesis

Starting with protein synthesis, we will first express the plasmid we designed in E. coli. We have streaked the bacteria on glycerol plates and will induce protein expression using IPTG. Following induction, we will harvest the cells and purify the expressed protein through affinity chromatography. To quantify the purified protein, we will perform a Western blot analysis, allowing us to confirm the expression and assess the yield of our target protein.

Induce Binding

Following the synthesis of our modified ABM, we will proceed to combine all components of our target complex.

First, we will induce the binding between albumin and ABM by placing them in the same environment. Next, we will synthesize the linker maleimide-PEG-OH through a thiol reaction, followed by its binding with Cefepime via esterification. The final product will be an albumin-ABM-linker-Cefepime complex, which can then be purified and prepared for administration to patients.

Binding Evaluation

Throughout each step of the binding process, we will implement a quantification strategy to confirm the success of our binding experiment. This will involve a two-step verification process: isolation followed by quantification.

We will utilize equilibrium dialysis to separate the bound products from the unbound materials. Subsequently, we will employ GC-MS to quantify the amount of bound products, allowing us to infer their affinity.

Discussion

Due to complications with plasmid design and shipping processes, we were unable to progress beyond the protein synthesis stage. If our design proves successful, we anticipate that cefepime will effectively attach to human serum albumin, and our modified ABM will enhance the overall affinity of the complex. We expect that cefepime will be retained in the bloodstream for extended periods, thereby reducing the frequency of drug administration and improving patients' quality of life.

Protocols