Quality control of inclusion bodies in Escherichia coli.
BACKGROUND
Bacterial inclusion bodies (IBs) are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous.


RESULTS
IBs of alpha-glucosidase of Saccharomyces cerevisiae after overproduction in Escherichia coli contain a large amount of (at least 12 different) major product fragments, as revealed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass-Spectrometry (MALDI-ToF MS) identification showed that these fragments contain either the N- or the C-terminus of the protein, therefore indicate that these IBs are at least partially created by proteolytic action. Expression of alpha-glucosidase in single knockout mutants for the major proteases ClpP, Lon, OmpT and FtsH which are known to be involved in the heat shock like response to production of recombinant proteins or to the degradation of IB proteins, clpP, lon, ompT, and ftsH did not influence the fragment pattern or the composition of the IBs. The quality of the IBs was also not influenced by the sampling time, cultivation medium (complex and mineral salt medium), production strategy (shake flask, fed-batch fermentation process), production strength (T5-lac or T7 promoter), strain background (K-12 or BL21), or addition of different protease inhibitors during IB preparation.


CONCLUSIONS
alpha-glucosidase is fragmented before aggregation, but neither by proteolytic action on the IBs by the common major proteases, nor during downstream IB preparation. Different fragments co-aggregate in the process of IB formation together with the full-length product. Other intracellular proteases than ClpP or Lon must be responsible for fragmentation. Reaggregation of protease-stable alpha-glucosidase fragments during in situ disintegration of the existing IBs does not seem to occur.
Jürgen
Britta
B
Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter Oulu, University of Oulu, FIN-90014 Oulu, Finland.
Breitenstein
Antje
A
Urlacher
Vlada
V
Büttner
Knut
K
Lin
Hongying
H
Hecker
Michael
M
Schweder
Thomas
T
Neubauer
Peter
P
D049069:ATP-Dependent Proteases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D049071:Endopeptidase Clp / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D049070:Protease La / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D011786:Quality Control; D012329:RNA, Bacterial / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000592:standards; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000592:standards; D012808:Sigma Factor / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism / Q000592:standards
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012808:Sigma Factor; C499588:ompT protein, E coli; C069567:sigma factor KatF protein, Bacteria; D000520:alpha-Glucosidases; D010447:Peptide Hydrolases; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; C483567:Lon protein, E coli; D049070:Protease La; C483572:ClpP protease, E coli; D049071:Endopeptidase Clp