Implementation of a Practical Teaching Course on Protein Engineering.
Luciana Calheiros GomesCarla FerreiraFilipe José MergulhãoPublished in: Biology (2022)
Protein Engineering is a highly evolved field of engineering aimed at developing proteins for specific industrial, medical, and research applications. Here, we present a practical teaching course to demonstrate fundamental techniques used to express, purify and analyze a recombinant protein produced in Escherichia coli -the enhanced green fluorescent protein (eGFP). The methodologies used for eGFP production were introduced sequentially over six laboratory sessions and included (i) bacterial growth, (ii) sonication (for cell lysis), (iii) affinity chromatography and dialysis (for eGFP purification), (iv) bicinchoninic acid (BCA) and fluorometry assays for total protein and eGFP quantification, respectively, and (v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for qualitative analysis. All groups were able to isolate the eGFP from the cell lysate with purity levels up to 72%. Additionally, a mass balance analysis performed by the students showed that eGFP yields up to 46% were achieved at the end of the purification process following the adopted procedures. A sensitivity analysis was performed to pinpoint the most critical steps of the downstream processing.
Keyphrases
- escherichia coli
- protein protein
- healthcare
- primary care
- binding protein
- single cell
- chronic kidney disease
- stem cells
- cell therapy
- mesenchymal stem cells
- systematic review
- risk assessment
- ms ms
- quantum dots
- high throughput
- staphylococcus aureus
- high resolution
- wastewater treatment
- recombinant human
- data analysis
- high speed
- end stage renal disease
- candida albicans
- high performance liquid chromatography