Multimeric intermediates in the pathway to the aggregated inclusion body state for P22 tailspike polypeptide chains.
The failure of newly synthesized polypeptide chains to reach the native conformation due to their accumulation as inclusion bodies is a serious problem in biotechnology. The critical intermediate at the junction between the productive folding and the inclusion body pathway has been previously identified for the P22 tailspike endorhamnosidase. We have been able to trap subsequent intermediates in the in vitro pathway to the aggregated inclusion body state. Nondenaturing gel electrophoresis identified a sequential series of multimeric intermediates in the aggregation pathway. These represent discrete species formed from noncovalent association of partially folded intermediates rather than aggregation of native-like trimeric species. Monomer, dimer, trimer, tetramer, pentamer, and hexamer states of the partially folded species were populated in the initial stages of the aggregation reaction. This methodology of isolating early multimers along the aggregation pathway was applicable to other proteins, such as the P22 coat protein and carbonic anhydrase II.
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Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge 02139, USA.
Wang
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King
J
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D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007181:Inclusion Bodies, Viral / Q000737:chemistry*; D007700:Kinetics; D015394:Molecular Structure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000378:metabolism; D014508:Urea; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D017376:Viral Tail Proteins
D014764:Viral Proteins; D017376:Viral Tail Proteins; D014508:Urea; D006026:Glycoside Hydrolases; C083268:tailspike protein, bacteriophage; D014357:Trypsin