Although　ribulose-1,5-bisphosphate　carboxylase/oxygenase　(RuBisCO)　has　been　obtained　from　green　tea　residue　mesophyll　cells　(TRMCs),　its　intact　release　has　not　yet　been　achieved.　To　release　RuBisCO,　this　study　employed　a　combination　or　sequential　treatments　using　urea,　β-mercaptoethanol,　sodium　dodecyl　sulfate　(SDS),　and　enzymes.　Factors　that　hindered　RuBisCO　release　from　TRMCs　were　investigated　through　SDS-PAGE　analysis,　protein　release　quantification,　and　electron　microscopy　techniques.　Alkali　treatment　of　TRMCs　at　95　°C　facilitated　protein　release,　while　also　causing　protein　modification　or　degradation.　Conversely,　the　combined　treatment　of　β-mercaptoethanol　with　urea　and/or　SDS　could　effectively　disrupt　the　disulfide　bonds,　hydrogen　bonds,　and/or　hydrophobic　interactions　within　the　cells,　leading　to　the　release　of　40%　or　more　of　the　proteins.　This　treatment　showed　strong　electrophoretic　bands　at　55　and　15　kDa,　indicating　that　RuBisCO　was　completely　released.　No　protein　was　released　during　the　treatment　with　SDS　and　pepsin/papain/alkaline　protease,　indicating　that　RuBisCO　was　hindered　by　the　presence　of　cellulose　and　hemicellulose.　Sequential　treatment　with　SDS　and　Viscozyme　L　dissolved　TRMC　lignocellulose　without　releasing　RuBisCO,　suggesting　the　low　solubility　of　RuBisCO.　Overall,　the　presence　of　lignocellulose　in　the　cell　wall　and　the　low　solubility　of　RuBisCO　were　identified　as　key　factors　hindering　its　release　from　the　TRMCs.　©　2024　American　Chemical　Society.