Intracellular ribosome display via SecM translation arrest as a selection for antibodies with enhanced cytosolic stability.
Ribosome display is a powerful approach for affinity and stability maturation of recombinant antibodies. However, since ribosome display is performed entirely in vitro, there are several limitations to this approach including technical challenges associated with: (i) efficiently expressing and stalling antibodies on ribosomes using cell-free translation mixtures; and (ii) folding of antibodies in buffers where the concentration and composition of factors varies from that found in the intracellular milieu. We have developed a novel method for intracellular ribosome display that takes advantage of the recently discovered Escherichia coli SecM translation arrest mechanism. Specifically, we provide the first evidence that the encoding mRNA of SecM-stalled heterologous proteins remains stably attached to ribosomes, thereby enabling creation of stalled antibody-ribosome-mRNA (ARM) complexes entirely inside of living cells. Since ARM complexes faithfully maintain a genotype-phenotype link between the arrested antibody and its encoding mRNA, we demonstrate that this method is ideally suited for isolating stability-enhanced single-chain variable fragment (scFv) antibodies that are efficiently folded and functional in the bacterial cytoplasm.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Lydia M
LM
Contreras-Martínez
Matthew P
MP
DeLisa
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042541:Intracellular Space; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010641:Phenotype; D014176:Protein Biosynthesis; D017510:Protein Folding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012995:Solubility; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D029968:Escherichia coli Proteins; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C497347:SecM protein, E coli; D014157:Transcription Factors