Dataset Information

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

ABSTRACT: A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatography of solubilized MMP-9, four protein peaks were obtained. However, so far, the different conformational protein species forming part of IBs have not been isolated and characterized. Therefore, with the aim to link the physicochemical characteristics of the isolated peaks with their biological activity, we set up a methodological approach that included dynamic light scattering (DLS), circular dichroism (CD), and spectrofluorometric analysis confirming the separation of subpopulations of conformers with specific characteristics. In protein purification procedures, the detailed analysis of the individual physicochemical properties and the biological activity of protein peaks separated by chromatographic techniques is a reliable source of information to select the best-fitted protein populations.

SUBMITTER: Carratala JV

PROVIDER: S-EPMC8001920 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

Carratalá Jose Vicente JV Gifre-Renom Laia L Roca-Pinilla Ramon R Villaverde Antonio A Arís Anna A Garcia-Fruitós Elena E Sánchez Julieta María JM Ferrer-Miralles Neus N

International journal of molecular sciences 20210316 6

A detailed workflow to analyze the physicochemical characteristics of mammalian matrix metalloproteinase (MMP-9) protein species obtained from protein aggregates (inclusion bodies-IBs) was followed. MMP-9 was recombinantly produced in the prokaryotic microbial cell factories Clearcoli (an engineered form of Escherichia coli) and Lactococcus lactis, mainly forming part of IBs and partially recovered under non-denaturing conditions. After the purification by affinity chromatog ...[more]

PMID: 33809594

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Project description:BackgroundImmobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically.ResultsWe here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein.ConclusionWe here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.

Project description: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.

Dataset Information

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

Publications

Selecting Subpopulations of High-Quality Protein Conformers among Conformational Mixtures of Recombinant Bovine MMP-9 Solubilized from Inclusion Bodies.

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