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. 2022 Mar 1;11(3):387.
doi: 10.3390/biology11030387.

Implementation of a Practical Teaching Course on Protein Engineering

Luciana C Gomes  1   2 , Carla Ferreira  3 , Filipe J Mergulhão  1   2
Affiliations

Implementation of a Practical Teaching Course on Protein Engineering

Luciana C Gomes et al. Biology (Basel). .

Abstract

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.

Keywords: green fluorescent protein; plasmid; protein purification; recombinant protein.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Outline of recombinant protein production and purification in Escherichia coli.
Figure 2
Figure 2
Plasmid pFM23 map. This harbours (i) a pMB1 origin of replication (ori), (ii) a repressor for the lac promoter (lacI), (iii) a transcriptional promoter from the T7 phage (T7 promoter), (iv) a lactose operator (lac operator), (v) an affinity purification tag (6 × His), (vi) a T7 transcriptional terminator (T7 terminator), (vii) a kanamycin resistance gene (KanR), and (viii) the eGFP gene (eGFP).
Figure 3
Figure 3
Student-generated growth curves of E. coli JM109(DE3) harbouring the pFM23 plasmid. After 180 min of incubation, IPTG was added to the culture medium.
Figure 4
Figure 4
Representative SDS-PAGE gel of the samples collected between cell lysis and protein dialysis (G to L). Lane M corresponds to Precision Plus Protein unstained standards (ref. 161-0363, Bio-Rad). The arrow indicates the bands corresponding to eGFP.
Figure 5
Figure 5
Process flow diagram and mass balance equations associated with eGFP purification.
See this image and copyright information in PMC

References

    1. Poluri K.M., Gulati K. Protein Engineering Techniques: Gateways to Synthetic Protein Universe. Springer; Singapore: 2017. Biotechnological and Biomedical Applications of Protein Engineering Methods; pp. 103–134.
    1. Gomes L.C., Mergulhão F.J. Production of Recombinant Proteins in Escherichia coli Biofilms: Challenges and Opportunities. In: Berhardt L.V., editor. Advances in Medicine and Biology. Volume 152 Nova Science Publishers; Hauppauge, NY, USA: 2019.
    1. Chalfie M., Tu Y., Euskirchen G., Ward W.W., Prasher D.C. Green fluorescent protein as a marker for gene expression. Science. 1994;263:802–805. doi: 10.1126/science.8303295. - DOI - PubMed
    1. Zacharias D.A., Tsien R.Y. Molecular biology and mutation of green fluorescent protein. Methods Biochem. Anal. 2006;47:83–120. - PubMed
    1. Stepanenko O.V., Stepanenko O.V., Kuznetsova I.M., Verkhusha V.V., Turoverov K.K. Beta-barrel scaffold of fluorescent proteins: Folding, stability and role in chromophore formation. Int. Rev. Cell. Mol. Biol. 2013;302:221–278. - PMC - PubMed

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