{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,13]],"date-time":"2026-01-13T22:10:53Z","timestamp":1768342253341,"version":"3.49.0"},"reference-count":33,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2022,3,1]],"date-time":"2022-03-01T00:00:00Z","timestamp":1646092800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FCT\/MCTES (PIDDAC)","award":["Base Funding - UIDB\/00511\/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy - LEPABE"],"award-info":[{"award-number":["Base Funding - UIDB\/00511\/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy - LEPABE"]}]},{"name":"FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionaliza\u00e7\u00e3o (POCI) and by national funds (PIDDAC) through FCT\/MCTES","award":["PTDC\/BII-BIO\/29589\/2017 - POCI-01-0145-FEDER-029589"],"award-info":[{"award-number":["PTDC\/BII-BIO\/29589\/2017 - POCI-01-0145-FEDER-029589"]}]},{"name":"FCT","award":["CEECIND\/01700\/2017"],"award-info":[{"award-number":["CEECIND\/01700\/2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>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\u2014the 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.<\/jats:p>","DOI":"10.3390\/biology11030387","type":"journal-article","created":{"date-parts":[[2022,3,1]],"date-time":"2022-03-01T08:17:03Z","timestamp":1646122623000},"page":"387","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Implementation of a Practical Teaching Course on Protein Engineering"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8992-1097","authenticated-orcid":false,"given":"Luciana","family":"Gomes","sequence":"first","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"given":"Carla","family":"Ferreira","sequence":"additional","affiliation":[{"name":"Department of Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5233-1037","authenticated-orcid":false,"given":"Filipe","family":"Mergulh\u00e3o","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,3,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Poluri, K.M., and Gulati, K. (2017). Biotechnological and Biomedical Applications of Protein Engineering Methods. Protein Engineering Techniques: Gateways to Synthetic Protein Universe, Springer.","DOI":"10.1007\/978-981-10-2732-1_5"},{"key":"ref_2","unstructured":"Berhardt, L.V. (2019). Production of Recombinant Proteins in Escherichia coli Biofilms: Challenges and Opportunities. Advances in Medicine and Biology, Nova Science Publishers."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"802","DOI":"10.1126\/science.8303295","article-title":"Green fluorescent protein as a marker for gene expression","volume":"263","author":"Chalfie","year":"1994","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1002\/0471739499.ch5","article-title":"Molecular biology and mutation of green fluorescent protein","volume":"47","author":"Zacharias","year":"2006","journal-title":"Methods Biochem. Anal."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/B978-0-12-407699-0.00004-2","article-title":"Beta-barrel scaffold of fluorescent proteins: Folding, stability and role in chromophore formation","volume":"302","author":"Stepanenko","year":"2013","journal-title":"Int. Rev. Cell. Mol. Biol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"338","DOI":"10.2174\/138920308785132668","article-title":"Fluorescent proteins as biomarkers and biosensors: Throwing color lights on molecular and cellular processes","volume":"9","author":"Stepanenko","year":"2008","journal-title":"Curr. Protein Pept. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/0378-1119(95)00685-0","article-title":"FACS-optimized mutants of the green fluorescent protein (GFP)","volume":"173","author":"Cormack","year":"1996","journal-title":"Gene"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1448","DOI":"10.1093\/nar\/gkh315","article-title":"Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: Library construction methods for directed evolution","volume":"32","author":"Neylon","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/0378-1119(95)00706-7","article-title":"Applications for green fluorescent protein (GFP) in the study of hostpathogen interactions","volume":"173","author":"Valdivia","year":"1996","journal-title":"Gene"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1038\/nbt0396-315","article-title":"Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling","volume":"14","author":"Crameri","year":"1996","journal-title":"Nat. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/S0091-679X(08)61963-9","article-title":"Flow Cytometric Analysis and FACS Sorting of Cells Based on GFP Accumulation","volume":"Volume 58","author":"Sullivan","year":"1998","journal-title":"Methods in Cell Biology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1016\/S0959-4388(00)00101-X","article-title":"Recent advances in technology for measuring and manipulating cell signals","volume":"10","author":"Zacharias","year":"2000","journal-title":"Curr. Opin. Neurobiol."},{"key":"ref_13","unstructured":"Berhardt, L.V. (2018). Applications of Green Fluorescent Protein in Biofilm Studies. Advances in Medicine and Biology, Nova Science Publishers."},{"key":"ref_14","first-page":"1236","article-title":"Analysis of factors affecting the periplasmic production of recombinant proteins in Escherichia coli","volume":"17","author":"Monteiro","year":"2007","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Gomes, L.C., Monteiro, G.A., and Mergulh\u00e3o, F.J. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by Escherichia coli Biofilms. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21020576"},{"key":"ref_16","unstructured":"Novagen (2005). pET System Manual, [11th ed.]. Available online: https:\/\/kirschner.med.harvard.edu\/files\/protocols\/Novagen_petsystem.pdf."},{"key":"ref_17","first-page":"1","article-title":"Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli","volume":"14","author":"Monteiro","year":"2004","journal-title":"J. Microbiol. Biotechnol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1186\/s12934-016-0437-3","article-title":"Recombinant pharmaceuticals from microbial cells: A 2015 update","volume":"15","author":"Mangues","year":"2016","journal-title":"Microb. Cell Factories"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1016\/S0958-1669(99)00003-8","article-title":"Recombinant protein expression in Escherichia coli","volume":"10","author":"Baneyx","year":"1999","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1385\/MB:12:1:25","article-title":"Expression and secretion of proteins in E. coli","volume":"12","author":"Pines","year":"1999","journal-title":"Mol. Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1016\/j.biotechadv.2004.11.003","article-title":"Recombinant protein secretion in Escherichia coli","volume":"23","author":"Summers","year":"2005","journal-title":"Biotechnol. Adv."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.jbiotec.2003.10.024","article-title":"Evaluation of bottlenecks in proinsulin secretion by Escherichia coli","volume":"109","author":"Taipa","year":"2004","journal-title":"J. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1475-2859-4-1","article-title":"Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli","volume":"4","author":"Mortensen","year":"2005","journal-title":"Microb. Cell Fact."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/S0076-6879(09)63026-3","article-title":"Affinity Chromatography: General Methods","volume":"Volume 463","author":"Burgess","year":"2009","journal-title":"Methods in Enzymology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/bs.mie.2014.11.003","article-title":"Purification of His-Tagged Proteins","volume":"Volume 559","author":"Lorsch","year":"2015","journal-title":"Methods in Enzymology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1021\/acsomega.7b01598","article-title":"Impact of an N-terminal Polyhistidine Tag on Protein Thermal Stability","volume":"3","author":"Booth","year":"2018","journal-title":"ACS Omega"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/0378-1119(85)90120-9","article-title":"Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors","volume":"33","author":"Vieira","year":"1985","journal-title":"Gene"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/0003-2697(85)90442-7","article-title":"Measurement of protein using bicinchoninic acid","volume":"150","author":"Smith","year":"1985","journal-title":"Anal. Biochem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1002\/bit.260380709","article-title":"Dynamics of induced CAT expression in E. coli","volume":"38","author":"Bentley","year":"1991","journal-title":"Biotechnol. Bioeng."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7136","DOI":"10.1016\/j.vaccine.2011.05.073","article-title":"Cloning and expression of protease ClpP from Streptococcus pneumoniae in Escherichia coli: Study of the influence of kanamycin and IPTG concentration on cell growth, recombinant protein production and plasmid stability","volume":"29","author":"Einsfeldt","year":"2011","journal-title":"Vaccine"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1961","DOI":"10.1021\/ed500482j","article-title":"Development and Validation of a Fast Procedure To Analyze Amoxicillin in River Waters by Direct-Injection LC\u2013MS\/MS","volume":"91","author":"Homem","year":"2014","journal-title":"J. Chem. Educ."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"420","DOI":"10.3389\/fbioe.2019.00420","article-title":"Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development","volume":"7","author":"Tripathi","year":"2019","journal-title":"Front. Bioeng. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Walker, J.M. (2009). SDS Polyacrylamide Gel Electrophoresis of Proteins. The Protein Protocols Handbook, Humana Press.","DOI":"10.1007\/978-1-59745-198-7_21"}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/11\/3\/387\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:30:02Z","timestamp":1760135402000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/11\/3\/387"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,1]]},"references-count":33,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2022,3]]}},"alternative-id":["biology11030387"],"URL":"https:\/\/doi.org\/10.3390\/biology11030387","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,1]]}}}