resumo
Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.
palavras-chave
ESCHERICHIA-COLI STRAINS; PICHIA-PASTORIS; MAMMALIAN-CELLS; CODON USAGE; RECOMBINANT EXPRESSION; FUNCTIONAL EXPRESSION; COUPLED RECEPTORS; OUTER-MEMBRANE; E. COLI; PURIFICATION
categoria
Biotechnology & Applied Microbiology
autores
Pedro, AQ; Queiroz, JA; Passarinha, LA
nossos autores
Augusto Pedro
Investigador JúniorProjectos
CICECO - Aveiro Institute of Materials (UID/CTM/50011/2019)
Towards the improvement of recombinant proteins bioprocessing using ionic liquids (IL2BioPro)
agradecimentos
The authors acknowledge the CICS-UBI projects Pest-OE/SAU/UI0709/2014, UID/Multi/00709/2013, and the program COMPETE, Pest-C/SAU/UI709/2011, financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER. CICS-UBI was also supported by FEDER funds through the POCI-COMPETE 2020 - Operational Programme Competitiveness and Internationalisation in Axis I -Strengthening research, technological development and innovation (Project POCI-01-0145-FEDER-007491). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. This work was also supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2019). The authors also acknowledge FCT for funding (Projects REFs: EXPL/BBB478/BQB/0960/2012 and POCI-01-0145-FEDER-030840). Augusto Q. Pedro acknowledges a doctoral fellowship (SFRH/BD/81222/2011) from FCT.