{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:56:36Z","timestamp":1790668596168,"version":"4.1.0"},"reference-count":129,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T00:00:00Z","timestamp":1790640000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Mol. Biosci."],"abstract":"<jats:p>\n                    Recombinant proteins are the workhorse of modern biochemical research, yet their use\n                    <jats:italic>in vitro<\/jats:italic>\n                    is routinely questioned on grounds of physiological relevance. Here we provide a structured guide to the strengths and limitations of recombinant protein studies, framed within the broader contrast between systemic (top-down) and reductionist (bottom-up) approaches to biological questions. We examine the main sources of concern. These are incorrect or incomplete folding, missing or aberrant post-translational modifications, the absence of native cofactors, the consequences of purification, and the lack of cellular crowding. Each of them is an experimentally measurable property rather than an assumption to be conceded. Against these we set the corresponding strengths: molecular-level resolution, well-defined interaction studies, and access to biophysical techniques that require purified material. The guide is organized around three practical tools, designed to be used in sequence: a comparison of expression hosts, a minimum checklist for validating a preparation before quantitative use, and a troubleshooting table for when a check fails. We argue that recombinant proteins are best understood not as a substitute for physiological studies but as a bridge between\n                    <jats:italic>in silico<\/jats:italic>\n                    ,\n                    <jats:italic>in vitro<\/jats:italic>\n                    , and\n                    <jats:italic>in vivo<\/jats:italic>\n                    research.\n                  <\/jats:p>","DOI":"10.3389\/fmolb.2026.1927411","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:19:46Z","timestamp":1790666386000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Understanding the strengths and limitations of recombinant protein studies"],"prefix":"10.3389","volume":"13","author":[{"given":"Daniele","family":"Dell\u2019Orco","sequence":"first","affiliation":[{"name":"Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona","place":["Verona, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karl-Wilhelm","family":"Koch","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Biochemistry Group, Carl von Ossietzky Universit\u00e4t Oldenburg","place":["Oldenburg, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,9,29]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"e3580","DOI":"10.21769\/BioProtoc.3580","article-title":"Quantitative determination of Ca2+-binding to Ca2+-sensor proteins by isothermal titration calorimetry","volume":"10","author":"Abbas","year":"2020","journal-title":"Bio Protoc."},{"key":"B2","doi-asserted-by":"publisher","first-page":"4374","DOI":"10.1021\/acs.biochem.9b00846","article-title":"Molecular recognition of rhodopsin kinase GRK1 and recoverin is tuned by switching Intra- and intermolecular electrostatic interactions","volume":"58","author":"Abbas","year":"2019","journal-title":"Biochemistry"},{"key":"B3","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1021\/ar50105a001","article-title":"Suicide enzyme inactivators","volume":"9","author":"Abeles","year":"1976","journal-title":"Acc. Chem. Res."},{"key":"B4","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"B5","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/bs.mie.2021.08.019","article-title":"Starting a new recombinant protein production project in Escherichia coli","volume":"659","author":"Aguilar Lucero","year":"2021","journal-title":"Methods Enzymol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"653","DOI":"10.1016\/s0956-5663(98)00020-7","article-title":"Surface plasmon resonance biosensors as a tool in antibody engineering","volume":"13","author":"Alfthan","year":"1998","journal-title":"Biosens. Bioelectron."},{"key":"B7","doi-asserted-by":"publisher","first-page":"371","DOI":"10.1007\/s00018-023-05022-0","article-title":"Recombinant protein delivery enables modulation of the phototransduction cascade in mouse retina","volume":"80","author":"Asteriti","year":"2023","journal-title":"Cell Mol. Life Sci."},{"key":"B8","doi-asserted-by":"publisher","first-page":"1488","DOI":"10.3390\/s16091488","article-title":"A guide to fluorescent protein FRET pairs","volume":"16","author":"Bajar","year":"2016","journal-title":"Sensors (Basel)"},{"key":"B9","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/nbt.1790","article-title":"Label-free quantification of membrane-ligand interactions using backscattering interferometry","volume":"29","author":"Baksh","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"861","DOI":"10.1038\/s41596-024-01079-8","article-title":"Biolayer interferometry for measuring the kinetics of protein\u2013protein interactions and nanobody binding","volume":"20","author":"Bates","year":"2025","journal-title":"Nat. Protoc."},{"key":"B11","doi-asserted-by":"publisher","first-page":"3720","DOI":"10.1016\/j.csbj.2021.06.033","article-title":"A hybrid stochastic\/deterministic model of single photon response and light adaptation in mouse rods","volume":"19","author":"Beelen","year":"2021","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"B12","doi-asserted-by":"publisher","first-page":"2625","DOI":"10.1038\/s41467-023-38328-5","article-title":"Improving de novo protein binder design with deep learning","volume":"14","author":"Bennett","year":"2023","journal-title":"Nat. Commun."},{"key":"B13","doi-asserted-by":"publisher","first-page":"123407","DOI":"10.1016\/j.ijbiomac.2023.123407","article-title":"Recombinant protein expression: challenges in production and folding related matters","volume":"233","author":"Beygmoradi","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"B14","doi-asserted-by":"publisher","first-page":"630551","DOI":"10.3389\/fbioe.2021.630551","article-title":"Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications","volume":"9","author":"Bhatwa","year":"2021","journal-title":"Front. Bioeng. Biotechnol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"757","DOI":"10.1038\/206757a0","article-title":"Structure of hen egg-white lysozyme. A three-dimensional Fourier synthesis at 2 Angstrom resolution","volume":"206","author":"Blake","year":"1965","journal-title":"Nature"},{"key":"B16","doi-asserted-by":"publisher","first-page":"882","DOI":"10.1038\/nprot.2016.044","article-title":"Integration and global analysis of isothermal titration calorimetry data for studying macromolecular interactions","volume":"11","author":"Brautigam","year":"2016","journal-title":"Nat. Protoc."},{"key":"B17","doi-asserted-by":"publisher","first-page":"2763","DOI":"10.3390\/ijms10062763","article-title":"Yeast two-hybrid, a powerful tool for systems biology","volume":"10","author":"Br\u00fcckner","year":"2009","journal-title":"Int. J. Mol. Sci."},{"key":"B18","doi-asserted-by":"publisher","first-page":"1086","DOI":"10.1038\/nm1001-1086","article-title":"Generating mice with targeted mutations","volume":"7","author":"Capecchi","year":"2001","journal-title":"Nat. Med."},{"key":"B19","doi-asserted-by":"publisher","first-page":"219","DOI":"10.1016\/0955-0674(94)90139-2","article-title":"Lipid modifications of G proteins","volume":"6","author":"Casey","year":"1994","journal-title":"Curr. Opin. Cell Biol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"719590","DOI":"10.3389\/fcell.2021.719590","article-title":"Post-translational modifications of the protein termini","volume":"9","author":"Chen","year":"2021","journal-title":"Front. Cell Dev. Biol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"27","DOI":"10.1146\/annurev-biochem-061516-045115","article-title":"Protein misfolding, amyloid formation, and human diesease: a summary of progress over the last decade","volume":"86","author":"Chiti","year":"2017","journal-title":"Annu. Rev. Biochem."},{"key":"B22","doi-asserted-by":"publisher","first-page":"551","DOI":"10.1038\/s41573-021-00195-4","article-title":"Kinase drug discovery 20 years after imatinib: progress and future directions","volume":"20","author":"Cohen","year":"2021","journal-title":"Nat. Rev. Drug Discov."},{"key":"B23","doi-asserted-by":"publisher","first-page":"127","DOI":"10.1007\/s00018-022-04165-w","article-title":"Alterations in calmodulin-cardiac ryanodine receptor molecular recognition in congenital arrhythmias","volume":"79","author":"Dal Cortivo","year":"2022","journal-title":"Cell Mol. Life Sci."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1100992","DOI":"10.3389\/fmolb.2022.1100992","article-title":"Calmodulin variants associated with congenital arrhythmia impair selectivity for ryanodine receptors","volume":"9","author":"Dal Cortivo","year":"2023","journal-title":"Front. Mol. Biosci."},{"key":"B25","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1007\/s00439-023-02629-y","article-title":"Impact of calmodulin missense variants associated with congenital arrhythmia on the thermal stability and the degree of unfolding","volume":"144","author":"Dal Cortivo","year":"2025","journal-title":"Hum. Genet."},{"key":"B26","doi-asserted-by":"publisher","first-page":"126568","DOI":"10.1016\/j.ijpharm.2026.126568","article-title":"Protein delivery to the eye: assessing therapeutic potential across inner and outer retina","volume":"690","author":"Dal Cortivo","year":"2026","journal-title":"Int. J. Pharm."},{"key":"B27","doi-asserted-by":"publisher","first-page":"106295","DOI":"10.1016\/j.pep.2023.106295","article-title":"A rapid procedure to generate stably transfected HEK293 suspension cells for recombinant protein manufacturing: yield improvements, bioreactor production and downstream processing","volume":"210","author":"Dash","year":"2023","journal-title":"Protein Expr. Purif."},{"key":"B28","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1126\/science.add2187","article-title":"Robust deep learning-based protein sequence design using ProteinMPNN","volume":"378","author":"Dauparas","year":"2022","journal-title":"Science"},{"key":"B30","doi-asserted-by":"publisher","first-page":"e201600272","DOI":"10.1002\/jbio.201600272","article-title":"Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET","volume":"11","author":"de Las Heras-Mart\u00ednez","year":"2018","journal-title":"J. Biophot."},{"key":"B31","doi-asserted-by":"publisher","first-page":"877","DOI":"10.1007\/s00449-024-03014-3","article-title":"Hybrid modeling of an ultracentrifugation process for separation of full and empty adeno-associated virus particles","volume":"47","author":"De-Luca","year":"2024","journal-title":"Bioprocess Biosyst. Eng."},{"key":"B32","doi-asserted-by":"publisher","first-page":"2982","DOI":"10.1021\/ac300213j","article-title":"Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device","volume":"84","author":"Dell'Orco","year":"2012","journal-title":"Anal. Chem."},{"key":"B33","doi-asserted-by":"publisher","first-page":"263","DOI":"10.1042\/BJ20110871","article-title":"A dynamic scaffolding mechanism for rhodopsin and transducin interaction in vertebrate vision","volume":"440","author":"Dell'Orco","year":"2011","journal-title":"Biochem. J."},{"key":"B34","doi-asserted-by":"publisher","first-page":"2390","DOI":"10.1021\/acschembio.6b00470","article-title":"Fingerprints of calcium-binding protein conformational dynamics monitored by Surface Plasmon Resonance","volume":"11","author":"Dell'Orco","year":"2016","journal-title":"ACS Chem. Biol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1232","DOI":"10.1039\/b908123b","article-title":"Network-level analysis of light adaptation in rod cells under normal and altered conditions","volume":"5","author":"Dell'Orco","year":"2009","journal-title":"Mol. Biosyst."},{"key":"B200","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1039\/b821580d","article-title":"Fast predictions of thermodynamics and kinetics of protein\u2013protein recognition from structures: from molecular design to systems biology","volume":"5","author":"Dell'Orco","year":"2009","journal-title":"Mol. Biosyst."},{"key":"B36","doi-asserted-by":"publisher","first-page":"7316","DOI":"10.1039\/c0cc02086a","article-title":"Quantitative detection of conformational transitions in a calcium sensor protein by surface plasmon resonance","volume":"46","author":"Dell'Orco","year":"","journal-title":"Chem. Commun. (Camb)"},{"key":"B37","doi-asserted-by":"publisher","first-page":"e10949","DOI":"10.1371\/journal.pone.0010949","article-title":"Modeling the time evolution of the nanoparticle-protein corona in a body fluid","volume":"5","author":"Dell'Orco","year":"","journal-title":"PLoS ONE"},{"key":"B38","doi-asserted-by":"publisher","first-page":"1008","DOI":"10.1016\/j.bpj.2016.02.001","article-title":"The Current revolution in Cryo-EM","volume":"110","author":"Egelman","year":"2016","journal-title":"Biophys. J."},{"key":"B39","doi-asserted-by":"publisher","first-page":"485","DOI":"10.1515\/BC.2006.064","article-title":"Protein aggregation in crowded environments","volume":"387","author":"Ellis","year":"2006","journal-title":"Biol. Chem."},{"key":"B40","doi-asserted-by":"publisher","first-page":"105569","DOI":"10.1016\/j.pep.2020.105569","article-title":"Production of membrane proteins in industry: the example of GPCRs","volume":"169","author":"Errey","year":"2020","journal-title":"Protein Expr. Purif."},{"key":"B41","doi-asserted-by":"publisher","first-page":"1081","DOI":"10.1038\/nm1001-1081","article-title":"The cultural mouse","volume":"7","author":"Evans","year":"2001","journal-title":"Nat. Med."},{"key":"B42","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1002\/jmr.1073","article-title":"Survey of the year 2009: applications of isothermal titration calorimetry","volume":"24","author":"Falconer","year":"2011","journal-title":"J. Mol. Recognit."},{"key":"B43","doi-asserted-by":"publisher","first-page":"e2901","DOI":"10.1002\/jmr.2901","article-title":"Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020","volume":"34","author":"Falconer","year":"2021","journal-title":"J. Mol. Recognit."},{"key":"B44","doi-asserted-by":"publisher","first-page":"4191","DOI":"10.1002\/j.1460-2075.1993.tb06103.x","article-title":"Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding","volume":"12","author":"Faurobert","year":"1993","journal-title":"EMBO J."},{"key":"B45","doi-asserted-by":"publisher","first-page":"286","DOI":"10.1016\/0168-9525(90)90012-u","article-title":"The two-hybrid system: an assay for protein-protein interactions","volume":"10","author":"Fields","year":"1994","journal-title":"Trends Genet."},{"key":"B46","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1016\/j.sbi.2016.01.001","article-title":"Using the folding landscapes of proteins to understand protein function","volume":"36","author":"Giri Rao","year":"2016","journal-title":"Curr. Opin. Struct. Biol."},{"key":"B47","doi-asserted-by":"crossref","first-page":"8647","DOI":"10.1016\/S0021-9258(18)31490-X","article-title":"Protein N-myristoylation","volume":"266","author":"Gordon","year":"1991","journal-title":"J. Biol. Chem."},{"key":"B48","doi-asserted-by":"publisher","first-page":"2043","DOI":"10.3390\/cells11132043","article-title":"Direct interaction of Avian cryptochrome 4 with a cone specific G-Protein","volume":"11","author":"G\u00f6rtemaker","year":"2022","journal-title":"Cells"},{"key":"B49","doi-asserted-by":"publisher","first-page":"2876","DOI":"10.1038\/nprot.2006.202","article-title":"Using circular dichroism spectra to estimate protein secondary structure","volume":"1","author":"Greenfield","year":"2006","journal-title":"Nat. Protoc."},{"key":"B50","doi-asserted-by":"publisher","first-page":"324","DOI":"10.1038\/nature10317","article-title":"Molecular chaperones in protein folding and proteostasis","volume":"475","author":"Hartl","year":"2011","journal-title":"Nature"},{"key":"B51","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1016\/j.ydbio.2006.08.065","article-title":"The immune gene repertoire encoded in the purple sea urchin genome","volume":"300","author":"Hibino","year":"2006","journal-title":"Dev. Biol."},{"key":"B52","doi-asserted-by":"publisher","first-page":"299","DOI":"10.1146\/annurev-biophys-032116-094545","article-title":"The radical-pair mechanism of magnetoreception","volume":"45","author":"Hore","year":"2016","journal-title":"Annu. Rev. Biophys."},{"key":"B53","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1186\/s13036-025-00562-4","article-title":"Insect cell expression system: advances in applications, engineering strategies, and bioprocess development","volume":"19","author":"Huang","year":"2025","journal-title":"J. Biol. Eng."},{"key":"B54","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1002\/(SICI)1099-1352(199905\/06)12:3<198::AID-JMR458>3.0.CO;2-9","article-title":"The use of biosensor technology for the engineering of antibodies and enzymes","volume":"12","author":"Huber","year":"1999","journal-title":"J. Mol. Recognit."},{"key":"B55","doi-asserted-by":"publisher","first-page":"1481","DOI":"10.1039\/c3mb70584f","article-title":"A comprehensive model of the phototransduction cascade in mouse rod cells","volume":"10","author":"Invergo","year":"2014","journal-title":"Mol. Biosyst."},{"key":"B56","doi-asserted-by":"publisher","first-page":"22253","DOI":"10.1038\/s41598-020-79226-w","article-title":"Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers","volume":"10","author":"Jankovics","year":"2020","journal-title":"Sci. Rep."},{"key":"B57","doi-asserted-by":"publisher","first-page":"1387","DOI":"10.1038\/nbt896","article-title":"FRET imaging","volume":"21","author":"Jares-Erijman","year":"2003","journal-title":"Nat. Biotechnol."},{"key":"B58","doi-asserted-by":"publisher","first-page":"100","DOI":"10.1016\/j.abb.2012.09.008","article-title":"Differential scanning calorimetry as a tool for protein folding and stability","volume":"531","author":"Johnson","year":"2013","journal-title":"Arch. Biochem. Biophys."},{"key":"B59","doi-asserted-by":"publisher","first-page":"10340","DOI":"10.1073\/pnas.91.22.10340","article-title":"Split ubiquitin as a sensor of protein interactions in vivo","volume":"91","author":"Johnsson","year":"1994","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B60","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly accurate protein structure prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"key":"B61","doi-asserted-by":"publisher","first-page":"1546","DOI":"10.1021\/acssensors.8b00361","article-title":"A highly compensated interferometer for biochemical analysis","volume":"3","author":"Kammer","year":"2018","journal-title":"ACS Sens."},{"key":"B62","doi-asserted-by":"publisher","first-page":"136","DOI":"10.1016\/j.ab.2005.09.034","article-title":"Analyzing a kinetic titration series using affinity biosensors","volume":"349","author":"Karlsson","year":"2006","journal-title":"Anal. Biochem."},{"key":"B108","doi-asserted-by":"publisher","first-page":"75","DOI":"10.1126\/science.309.5731.75","article-title":"What don\u2019t we know?","volume":"309","author":"Kennedy","year":"2005","journal-title":"Science"},{"key":"B63","doi-asserted-by":"publisher","first-page":"2411","DOI":"10.1007\/s00253-020-10402-8","article-title":"Fusion tags to enhance heterologous protein expression","volume":"104","author":"Ki","year":"2020","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"B64","doi-asserted-by":"publisher","first-page":"323","DOI":"10.1146\/annurev-biochem-060208-092442","article-title":"Molecular chaperone functions in protein folding and proteostasis","volume":"82","author":"Kim","year":"2013","journal-title":"Annu. Rev. Biochem."},{"key":"B65","doi-asserted-by":"publisher","first-page":"101248","DOI":"10.1016\/j.preteyeres.2024.101248","article-title":"Pluripotent stem cell-derived models of retinal disease: elucidating pathogenesis, evaluating novel treatments, and estimating toxicity","volume":"100","author":"Kurzawa-Akanbi","year":"2024","journal-title":"Prog. Retin Eye Res."},{"key":"B66","doi-asserted-by":"publisher","first-page":"681","DOI":"10.1194\/jlr.R600002-JLR200","article-title":"Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I","volume":"47","author":"Lane","year":"2006","journal-title":"J. Lipid Res."},{"key":"B67","doi-asserted-by":"publisher","first-page":"408","DOI":"10.2174\/138920109788488851","article-title":"Suppression of protein aggregation by L-arginine","volume":"10","author":"Lange","year":"2009","journal-title":"Curr. Pharm. Biotechnol."},{"key":"B68","doi-asserted-by":"publisher","first-page":"1468","DOI":"10.3390\/biom15101468","article-title":"Proximity ligation assay: from a foundational principle to a versatile platform for molecular and translational research","volume":"15","author":"Li","year":"2025","journal-title":"Biomolecules"},{"key":"B69","doi-asserted-by":"publisher","first-page":"1408","DOI":"10.1038\/aps.2015.82","article-title":"A new trend to determine biochemical parameters by quantitative FRET assays","volume":"36","author":"Liao","year":"2015","journal-title":"Acta Pharmacol. Sin."},{"key":"B70","doi-asserted-by":"publisher","first-page":"497","DOI":"10.1016\/s0958-1669(98)80035-9","article-title":"Advances in refolding of proteins produced in E. coli","volume":"9","author":"Lilie","year":"1998","journal-title":"Curr. Opin. Biotechnol."},{"key":"B71","doi-asserted-by":"publisher","first-page":"1123","DOI":"10.1126\/science.ade2574","article-title":"Evolutionary-scale prediction of atomic-level protein structure with a language model","volume":"379","author":"Lin","year":"2023","journal-title":"Science"},{"key":"B72","doi-asserted-by":"publisher","first-page":"673","DOI":"10.1007\/s40259-026-00783-z","article-title":"Recombinant Protein drugs: a 2025 update","volume":"40","author":"L\u00f3pez-Laguna","year":"2026","journal-title":"BioDrugs"},{"key":"B73","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1038\/s41586-022-04456-z","article-title":"The road to fully programmable protein catalysis","volume":"606","author":"Lovelock","year":"2022","journal-title":"Nature"},{"key":"B74","doi-asserted-by":"publisher","first-page":"M110.004978","DOI":"10.1074\/mcp.M110.004978","article-title":"Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material","volume":"10","author":"Lundberg","year":"2011","journal-title":"Mol. Cell. Proteom."},{"key":"B29","doi-asserted-by":"publisher","first-page":"21","DOI":"10.1186\/s12934-025-02646-8","article-title":"Recent advances in recombinant production of soluble proteins in E. coli","volume":"24","author":"de Marco","year":"2025","journal-title":"Microb. Cell Factories"},{"key":"B75","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1146\/annurev.ge.20.120186.000501","article-title":"The evolution of multigene families: human haptoglobin genes","volume":"20","author":"Maeda","year":"1986","journal-title":"Annu. Rev. Genet."},{"key":"B76","doi-asserted-by":"publisher","first-page":"367","DOI":"10.1016\/j.ijbiomac.2017.04.025","article-title":"An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding","volume":"102","author":"Mamipour","year":"2017","journal-title":"Int. J. Biol. Macromol."},{"key":"B77","doi-asserted-by":"publisher","first-page":"6653","DOI":"10.1093\/hmg\/ddv370","article-title":"Two retinal dystrophy-associated missense mutations in GUCA1A with distinct molecular properties result in a similar aberrant regulation of the retinal guanylate cyclase","volume":"24","author":"Marino","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"B78","doi-asserted-by":"publisher","first-page":"11773","DOI":"10.1039\/c7nr03288a","article-title":"CaF2 nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies","volume":"9","author":"Marino","year":"2017","journal-title":"Nanoscale"},{"key":"B79","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1016\/0022-2836(68)90205-2","article-title":"Solvent content of protein crystals","volume":"33","author":"Matthews","year":"1968","journal-title":"J. Mol. Biol."},{"key":"B80","doi-asserted-by":"publisher","first-page":"529","DOI":"10.2174\/0929866526666191112152646","article-title":"A comprehensive guide to the commercial baculovirus expression vector systems for recombinant protein production","volume":"27","author":"Mishra","year":"2020","journal-title":"Protein Pept. Lett."},{"key":"B81","doi-asserted-by":"publisher","first-page":"6705","DOI":"10.1073\/pnas.95.12.6705","article-title":"Expressed protein ligation: a general method for protein engineering","volume":"95","author":"Muir","year":"1998","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B82","doi-asserted-by":"publisher","first-page":"279","DOI":"10.1002\/(SICI)1099-1352(199909\/10)12:5<279::AID-JMR473>3.0.CO;2-3","article-title":"Improving biosensor analysis","volume":"12","author":"Myszka","year":"1999","journal-title":"J. Mol. Recognit."},{"key":"B83","doi-asserted-by":"publisher","first-page":"e7","DOI":"10.1017\/S0033583526100134","article-title":"Energy landscapes in molecular biology: history, principles, and perspectives","volume":"59","author":"Nussinov","year":"2026","journal-title":"Q. Rev. Biophys."},{"key":"B84","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1007\/s00253-017-8623-8","article-title":"Guidelines to reach high-quality purified recombinant proteins","volume":"102","author":"Oliveira","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"B85","doi-asserted-by":"publisher","first-page":"184","DOI":"10.7324\/JAPS.2021.110824","article-title":"Advances in the science and technology of insulin delivery: a review","volume":"11","author":"Olorunsola","year":"2021","journal-title":"J. Appl. Pharm. Sci."},{"key":"B86","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1016\/j.tibs.2024.12.012","article-title":"Protein N-terminal modifications: molecular machineries and biological implications","volume":"50","author":"\u00d8ye","year":"2025","journal-title":"Trends Biochem. Sci."},{"key":"B87","doi-asserted-by":"publisher","first-page":"739","DOI":"10.1126\/science.289.5480.739","article-title":"Crystal structure of rhodopsin: a G protein-coupled receptor","volume":"289","author":"Palczewski","year":"2000","journal-title":"Science"},{"key":"B88","doi-asserted-by":"publisher","first-page":"343","DOI":"10.1016\/s0092-8674(85)80004-0","article-title":"Transgenic mice","volume":"41","author":"Palmiter","year":"1985","journal-title":"Cell"},{"key":"B89","doi-asserted-by":"publisher","first-page":"23830","DOI":"10.1074\/jbc.M600257200","article-title":"Ca2+ and Mg2+ binding properties of GCAP-1. Evidence that Mg2+-bound form is the physiological activator of photoreceptor guanylyl cyclase","volume":"281","author":"Peshenko","year":"2006","journal-title":"J. Biol. Chem."},{"key":"B90","doi-asserted-by":"publisher","first-page":"21645","DOI":"10.1074\/jbc.M702368200","article-title":"Activation and inhibition of photoreceptor guanylyl cyclase by guanylyl cyclase activating protein 1 (GCAP-1): the functional role of Mg2+\/Ca2+ exchange in EF-hand domains","volume":"282","author":"Peshenko","year":"2007","journal-title":"J. Biol. Chem."},{"key":"B91","doi-asserted-by":"publisher","first-page":"102690","DOI":"10.1016\/j.cbpa.2026.102690","article-title":"Genetic code expansion: bridging synthetic biology precision and cellular complexity to decipher molecular mechanisms of diseases","volume":"93","author":"Phromkrasae","year":"2026","journal-title":"Curr. Opin. Chem. Biol."},{"key":"B92","first-page":"3","article-title":"Biophysical approaches to the study of G-protein structure and function","volume-title":"Chapter 1 in Signal Transduction in the Retina","author":"Preininger","year":"2008"},{"key":"B93","doi-asserted-by":"publisher","first-page":"549","DOI":"10.1038\/nature10361","article-title":"Crystal structure of the \u03b22 adrenergic receptor-Gs protein complex","volume":"477","author":"Rasmussen","year":"2011","journal-title":"Nature"},{"key":"B94","doi-asserted-by":"publisher","first-page":"300","DOI":"10.1002\/jmr.862","article-title":"Survey of the year 2006 commercial optical biosensor literature","volume":"20","author":"Rich","year":"2007","journal-title":"J. Mol. Recognit."},{"key":"B95","doi-asserted-by":"publisher","first-page":"172","DOI":"10.3389\/fmicb.2014.00172","article-title":"Recombinant protein expression in Escherichia coli: advances and challenges","volume":"5","author":"Rosano","year":"2014","journal-title":"Front. Microbiol."},{"key":"B96","doi-asserted-by":"publisher","first-page":"1412","DOI":"10.1002\/pro.3668","article-title":"New tools for recombinant protein production in Escherichia coli: a 5-year update","volume":"28","author":"Rosano","year":"2019","journal-title":"Protein Sci."},{"key":"B97","doi-asserted-by":"publisher","first-page":"198","DOI":"10.1016\/j.ymeth.2008.09.003","article-title":"Isothermal titration calorimetry of RNA","volume":"47","author":"Salim","year":"2009","journal-title":"Methods"},{"key":"B98","doi-asserted-by":"publisher","first-page":"729","DOI":"10.1038\/s41580-020-00294-x","article-title":"Global view of human protein glycosylation pathways and functions","volume":"21","author":"Schjoldager","year":"2020","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"B99","doi-asserted-by":"publisher","first-page":"541","DOI":"10.1146\/annurev.biophys.26.1.541","article-title":"Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules","volume":"26","author":"Schuck","year":"1997","journal-title":"Annu. Rev. Biophys. Biomol. Struct."},{"key":"B100","doi-asserted-by":"publisher","first-page":"102572","DOI":"10.1016\/j.xpro.2023.102572","article-title":"A concise guide to choosing suitable gene expression systems for recombinant protein production","volume":"4","author":"Sch\u00fctz","year":"2023","journal-title":"Star. Protoc."},{"key":"B101","doi-asserted-by":"publisher","first-page":"561","DOI":"10.1038\/nature11499","article-title":"Skin shedding and tissue regeneration in African spiny mice (Acomys)","volume":"489","author":"Seifert","year":"2012","journal-title":"Nature"},{"key":"B102","doi-asserted-by":"publisher","first-page":"1549585","DOI":"10.3389\/fphys.2025.1549585","article-title":"AAV-mediated transduction of songbird retina","volume":"16","author":"Seth","year":"2025","journal-title":"Front. Physiol."},{"key":"B103","doi-asserted-by":"publisher","first-page":"905","DOI":"10.1038\/nmeth819","article-title":"A guide to choosing fluorescent proteins","volume":"2","author":"Shaner","year":"2005","journal-title":"Nat. Methods"},{"key":"B104","doi-asserted-by":"publisher","first-page":"4159","DOI":"10.1002\/bit.27912","article-title":"In vivo cleavage of solubility tags as a tool to enhance the levels of soluble recombinant proteins in Escherichia coli","volume":"118","author":"Silva","year":"2021","journal-title":"Biotechnol. Bioeng."},{"key":"B105","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1038\/s41576-019-0186-3","article-title":"Cell-free gene expression: an expanded repertoire of applications","volume":"21","author":"Silverman","year":"2020","journal-title":"Nat. Rev. Genet."},{"key":"B106","doi-asserted-by":"publisher","first-page":"102697","DOI":"10.1016\/j.sbi.2023.102697","article-title":"Conformational entropy in molecular recognition of intrinsically disordered proteins","volume":"83","author":"Skriver","year":"2023","journal-title":"Curr. Opin. Struct. Biol."},{"key":"B107","doi-asserted-by":"publisher","first-page":"181","DOI":"10.2337\/diacare.5.2.s181","article-title":"Human insulin of recombinant DNA origin: clinical potential","volume":"5","author":"Skyler","year":"1982","journal-title":"Diabetes Care"},{"key":"B109","doi-asserted-by":"publisher","first-page":"135","DOI":"10.1038\/nmeth.f.202","article-title":"Protein production and purification","volume":"5","author":"Gr\u00e4slund","year":"2008","journal-title":"Nat. Methods"},{"key":"B110","doi-asserted-by":"publisher","first-page":"e53568","DOI":"10.3791\/53568","article-title":"High yield expression of recombinant human proteins with the transient transfection of HEK293 cells in suspension","volume":"106","author":"Subedi","year":"2015","journal-title":"J. Vis. Exp."},{"key":"B111","doi-asserted-by":"publisher","first-page":"6756","DOI":"10.1002\/chem.201402146","article-title":"Conformational changes in calcium-sensor proteins under molecular crowding conditions","volume":"20","author":"Sulmann","year":"2014","journal-title":"Chemistry"},{"key":"B112","doi-asserted-by":"publisher","first-page":"45515","DOI":"10.1038\/srep45515","article-title":"Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry","volume":"7","author":"Sulmann","year":"2017","journal-title":"Sci. Rep."},{"key":"B113","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1110\/ps.03288104","article-title":"Protein folding by the effects of macromolecular crowding","volume":"13","author":"Tokuriki","year":"2004","journal-title":"Protein Sci."},{"key":"B114","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1146\/annurev.bi.57.070188.000441","article-title":"The biology and enzymology of eukaryotic protein acylation","volume":"57","author":"Towler","year":"1988","journal-title":"Annu. Rev. Biochem."},{"key":"B115","doi-asserted-by":"publisher","first-page":"6589","DOI":"10.1021\/cr400525m","article-title":"Classification of intrinsically disordered regions and proteins","volume":"114","author":"van der Lee","year":"2014","journal-title":"Chem. Rev."},{"key":"B116","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1016\/j.jsb.2010.05.005","article-title":"Biophysical characterization of recombinant proteins: a key to higher structural genomics success","volume":"172","author":"Vedadi","year":"2010","journal-title":"J. Struct. Biol."},{"key":"B117","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1093\/hmg\/ddw374","article-title":"Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1","volume":"26","author":"Vocke","year":"2017","journal-title":"Hum. Mol. Genet."},{"key":"B118","doi-asserted-by":"publisher","first-page":"1790","DOI":"10.1126\/science.298.5599.1790","article-title":"N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli","volume":"298","author":"Wacker","year":"2002","journal-title":"Science"},{"key":"B119","doi-asserted-by":"publisher","first-page":"1722","DOI":"10.1038\/s41587-022-01582-x","article-title":"Biopharmaceutical benchmarks 2022","volume":"40","author":"Walsh","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"B120","doi-asserted-by":"publisher","first-page":"110","DOI":"10.1038\/nrm.2015.11","article-title":"Protein prenylation: unique fats make their mark on biology","volume":"17","author":"Wang","year":"2016","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"B121","doi-asserted-by":"publisher","first-page":"1089","DOI":"10.1038\/s41586-023-06415-8","article-title":"De novo design of protein structure and function with RFdiffusion","volume":"620","author":"Watson","year":"2023","journal-title":"Nature"},{"key":"B122","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1167\/tvst.13.3.9","article-title":"Long-Term Porcine Retina explants as an alternative to In Vivo experimentation","volume":"13","author":"Weller","year":"2024","journal-title":"Transl. Vis. Sci. Technol."},{"key":"B123","doi-asserted-by":"publisher","first-page":"883","DOI":"10.1194\/jlr.R600004-JLR200","article-title":"Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras","volume":"47","author":"Wright","year":"2006","journal-title":"J. Lipid Res."},{"key":"B124","doi-asserted-by":"publisher","first-page":"535","DOI":"10.1038\/s41586-021-03618-9","article-title":"Magnetic sensitivity of cryptochrome 4 from a migratory songbird","volume":"594","author":"Xu","year":"2021","journal-title":"Nature"},{"key":"B125","doi-asserted-by":"publisher","first-page":"1139","DOI":"10.1038\/s41586-026-10328-7","article-title":"The past, present and future of de novo protein design","volume":"652","author":"Yang","year":"2026","journal-title":"Nature"},{"key":"B126","doi-asserted-by":"publisher","first-page":"14541","DOI":"10.1021\/bi0352365","article-title":"Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: insertion of 3-nitrotyrosine at residue 356 as a probe of the radical initiation process","volume":"42","author":"Yee","year":"2003","journal-title":"Biochemistry"},{"key":"B127","doi-asserted-by":"publisher","first-page":"20.2.1","DOI":"10.1002\/cpps.31","article-title":"Measuring protein interactions by optical biosensors","volume":"88","author":"Zhao","year":"2017","journal-title":"Curr. Protoc. Protein Sci."},{"key":"B128","doi-asserted-by":"publisher","first-page":"375","DOI":"10.1146\/annurev.biophys.37.032807.125817","article-title":"Macromolecular crowding and confinement: biochemical, biophysical, and potential physiological consequences","volume":"37","author":"Zhou","year":"2008","journal-title":"Annu. Rev. Biophys."}],"container-title":["Frontiers in Molecular Biosciences"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmolb.2026.1927411\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:19:52Z","timestamp":1790666392000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fmolb.2026.1927411\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,29]]},"references-count":129,"alternative-id":["10.3389\/fmolb.2026.1927411"],"URL":"https:\/\/doi.org\/10.3389\/fmolb.2026.1927411","relation":{},"ISSN":["2296-889X"],"issn-type":[{"value":"2296-889X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,29]]},"article-number":"1927411"}}