Molecular characterization of beta-lactamase inclusion bodies produced in Escherichia coli. 1. Composition.
We have determined the macromolecular composition of inclusion bodies formed by overexpressing beta-lactamase from three different expression systems as a function of the growth conditions. The inclusion bodies were purified by differential gradient centrifugation and detergent extraction. Both the expression system and the growth conditions were shown to have a pronounced effect on inclusion body composition. Specifically, contaminating polypeptides ranged from less than 5% to over 50% of the total protein content. Phospholipids composed 0.5-13% of the inclusion bodies. Nucleic acids represented a minor impurity for both cytoplasmic and periplasmic inclusion bodies. Cytoplasmic inclusion bodies of the mature beta-lactamase had the lowest amount of impurities, irrespective of the growth conditions. On the other hand, large amounts of outer membrane proteins and phospholipids were observed in periplasmic inclusion bodies from cells grown at basic pH. Our results show that, at least under some growth conditions, protein aggregation in vivo is highly specific, and the presence of contaminating proteins in inclusion bodies is due to incomplete purification following cell lysis.
Valax
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Department of Chemical Engineering, University of Texas, Austin 78712.
Georgiou
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D001426:Bacterial Proteins / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000737:chemistry / Q000201:enzymology*; D046911:Macromolecular Substances; D009696:Nucleic Acids / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D012995:Solubility; D013696:Temperature; D001618:beta-Lactamases / Q000032:analysis* / Q000096:biosynthesis*
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D009696:Nucleic Acids; D010743:Phospholipids; D001618:beta-Lactamases