Localization of Functional Polypeptides in Bacterial Inclusion Bodies

Abstract

Bacterial inclusion bodies, while showing intriguing amyloid-like features, such as a ␤-sheet-based intermolecular organization, binding to amyloid-tropic dyes, and origin in a sequence-selective deposition process, hold an important amount of native-like secondary structure and significant amounts of functional polypeptides. The aggregation mechanics supporting the occurrence of both misfolded and properly folded protein is controversial. Single polypeptide chains might contain both misfolded stretches driving aggregation and properly folded protein domains that, if embracing the active site, would account for the biological activities displayed by inclusion bodies. Alternatively, soluble, functional polypeptides could be surface adsorbed by interactions weaker than those driving the formation of the intermolecular ␤-sheet architecture. To explore whether the fraction of properly folded active protein is a natural component or rather a mere contaminant of these aggregates, we have explored their localization by image analysis of inclusion bodies formed by green fluorescent protein. Since the fluorescence distribution is not homogeneous and the core of inclusion bodies is particularly rich in active protein forms, such protein species cannot be passively trapped components and their occurrence might be linked to the reconstruction dynamics steadily endured in vivo by such bacterial aggregates. Intriguingly, even functional protein species in inclusion bodies are not excluded from the interface with the solvent, probably because of the porous structure of these particular protein aggregates.Procedures for in vitro protein refolding are under continuous development (22,32,35), since many proteins of industrial or pharmacological interest are produced in recombinant microorganisms, especially bacteria, as insoluble aggregates called inclusion bodies (IBs) (38). Recent insights into the structure and physiology of bacterial IBs have revealed that at least a significant fraction of the embedded protein occurs in a properly folded native-like form (36) and that for aggregates formed by enzymes, this fact is reflected by the occurrence of enzymatic activity associated with these particles (16,34,39). While for hormones or other drugs to be used in vivo, in vitro solubilization of IBs and refolding of IB proteins would still be required to allow their proper use (27), enzymes to be used in bioprocesses could be employed straight after production, skipping any refolding step. This is particularly appealing since the specific activity found in IB enzymes, although variable when comparing different protein species, is not dramatically different from that exhibited by the soluble counterparts (15, 16), and on the other hand, refolding procedures render yields of active protein that are usually far from 100%.Apart from the obvious potential of enzyme IBs as catalyzers, the occurrence of properly folded, active enzymes poses intriguing structural questions. The conformational background sustaining the IB molecular structure l...

The effects of protein solubility on the RNA Integrity Number (RIN) for recombinant Escherichia coli

“…Flow cytometry analysis of the induced VP1GFP cultures showed the fluorescent intensity increased approximately 8-fold at 60-minutes post-induction and approximately 30-fold at 3.5-hours post-induction. Fluorescence microscopy showed the VP1GFP protein was localized in the flagella end of the cells, which was also reported and characterized by Dr. Villaverde's research group as inclusion bodies [57]. The induction levels for both GFPCAT and VP1GFP were similar under the experimental conditions, which was expected for these very similar plasmid constructs.…”

“…The mean signal acquisition from 50,000 cells was used to characterize sample intensity. VP1GFP inclusion bodies were confirmed by fluorescence microscopy (Nikon Ti, 60X TIRF oil) [57]. …”

Optimizing Protein Stability In Vivo

“…However, simple proteolytic destruction of the test protein, as might be expected for poorly folded proteins, can relieve the interference and lead to false positive results. Since a protein's solubility is not necessarily related to its activity or thermodynamic stability (Famm et al, 2008; Garcia-Fruitos et al, 2007), it is not possible to separate which of these phenomena causes the enhanced antibiotic resistance in our examples; however, the results highlight the potential of the tripartite system as a means to select variants of proteins that are expressed in soluble form and are thus easier to produce, handle, and characterize.…”

Surface Cell Growth Engineering Assisted by a Novel Bacterial Nanomaterial

“…[3] While in the past IBs were believed to be formed by unfolded or largely misfolded polypeptide chains and therefore biologically inert, [5] recent insights show them to be constituted by folded and biofunctional protein species, [6] whose presence is allowed by a particular amyloid-like organization. [7][8][9] Therefore, IBs formed by enzymes such as b-galactosidase, D-amino acid oxidase, maltodextrin phosphorylase, sialic acid aldolase, and polyphosphate kinase [8,[10][11][12][13] can be used as catalysts in different processes. On the other hand, the in vivo formation of IBs is regulated by several cellular genes (mainly encoding proteases and chaperones), which makes the genetic manipulation of their nanometer-scale properties feasible.…”