Quality control of inclusion bodies in Escherichia coli

Abstract

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.

Figure 1

Profile of a glucose limited fed-batch fermentation with constant glucose feed rate of E. coli RB791 overproducing the α-glucosidase after induction of the tac-promoter with IPTG. Data from two different independent cultivations are shown. Cell dry weight (CDW, triangles, squares), α-glucosidase (bars, S.D. of quantified samples).

Figure 2

2D PAGE analysis of the IB protein fraction of the overproduced α-glucosidase (GLUCP1) 3 hours after induction with IPTG. MALDI-ToF MS analysis and N-terminal sequencing revealed that the majority of the detected protein spot can be assigned to the α-glucosidase (GLUCP1-1 to GLUCP1-12).

Figure 3

Sequence coverage of the different peptides of α-glucosidase protein fragments identified by MALDI-ToF MS or N-terminal sequencing (GLUCP1-1 to GLUCP1-12). The indentified peptides are serially numbered and marked with brackets which denote the individual fragments of the detected spots (see also Table 1, Additional file 1).

Figure 4

2D PAGE of the IB fraction of the E. coli strain deficient in rpoS and clpP overproducing the α-glucosidase.

Figure 5

2D PAGE of the IB fraction of an E. coli Lon deficient strain overproducing the α-glucosidase.

Figure 6

2D PAGE of the IB fraction of an E. coli OmpT deficient strain overproducing the α-glucosidase.

Figure 7

2D PAGE of the IB fraction of an E. coli FtsH deficient strain overproducing the α-glucosidase.

Figure 8

Northern Blot analyses (A-C) of the α-glucosidase transcript with different oligonucleotide probes. Analyses were performed with samples from E. coli RB791 pKK177glucC pUBS520 collected from batch cultivations 60 min after induction. Different probes were tested, covering the glucC mRNA sequence from position 374 -to 399 (A), 1160 to 1185 (B), and from position 1580 to 1605 (C). The right side of the figure (D) indicates schematically the different locations of the analysed mRNA fragments in relation to the glucC sequence. The probe locations are indicated by red bars. Non-translated 5' regions of the mRNA transcript are shown as a dashed line.