Active protein aggregates induced by terminally attached self-assembling peptide ELK16 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

In recent years, it has been gradually realized that bacterial inclusion bodies (IBs) could be biologically active. In particular, several proteins including green fluorescent protein, β-galactosidase, β-lactamase, alkaline phosphatase, D-amino acid oxidase, polyphosphate kinase 3, maltodextrin phosphorylase, and sialic acid aldolase have been successfully produced as active IBs when fused to an appropriate partner such as the foot-and-mouth disease virus capsid protein VP1, or the human β-amyloid peptide Aβ42(F19D). As active IBs may have many attractive advantages in enzyme production and industrial applications, it is of considerable interest to explore them further.     

Immobilization of fuculose-1-phosphate aldolase from E. coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from E. coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80-90% and FucA retained activity on immobilized derivative of 10-20% can be achieved when pH 10, 20°C and 200 µmoles cm^-3 of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme.


A highly loaded derivative of immobilized FucA (65 AU cm^-3 of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)- alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.     

Immobilization of fuculose-1-phosphate aldolase from E. coli to glyoxal-agarose gels by multipoint covalent attachment

Recombinant fuculose 1-phosphate aldolase (FucA) from E. coli has been immobilized by multipoint covalent attachment to glyoxal-agarose gels. Experiments, varying the main parameters that control the immobilization process (surface density of aldehyde groups, temperature, pH), were carried out. An immobilization yield of 80–90% and FucA retained activity on immobilized derivative of 10–20% can be achieved when pH?10, 20°C and 200?µmoles?cm^?3 of aldehyde groups was used. The observed activity loss in the immobilization process might be related to the fact that the complex quaternary structure of the enzyme could not be maintained. A short contact-time enzyme support is required to obtain high ratio of active to total immobilized enzyme.

A highly loaded derivative of immobilized FucA (65?AU?cm^?3 of support) has been prepared to use in aldol condensation reactions. Reactions catalyzed by these aldolases involve the use of non-conventional media because of substrate solubility. For instance, the condensation of dihydroxyacetone phosphate (DHAP) and Z-amino-propanal, Z-(R)-alaninal and Z-(S)- alaninal in highly concentrated water-in-oil emulsions gave synthetic yields of 40, 25 and 29% respectively.     

Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli

An unsteady, unstructured, unsegregated and based on first principles mathematical model has been proposed to describe IPTG (isopropyl-β-d-tiogalactopiranoside) transport in induced fed-batch cultures of E. coli M15 ΔglyA [pQEαβrham] [pREP4] producing rhamnulose 1-phosphate aldolase (RhuA). The model predicts extracellular and intracellular IPTG concentration. Experimental extracellular IPTG concentrations under different operational conditions were obtained by HPLC–MS analysis. These experimental data were used to fit the parameters of the model. The model was also able to predict the experimental behavior of two different E. coli strains producing fuculose 1-phosphate aldolase (FucA). IPTG transport to cells was the contribution of three processes: a diffusion process, and two active processes (one non-specific and another specific).     

Techno-economic evaluation of an inclusion body solubilization and recombinant protein refolding process

Expression of recombinant proteins in Escherichia coli is normally accompanied by the formation of inclusion bodies (IBs). To obtain the protein product in an active (native) soluble form, the IBs must be first solubilized, and thereafter, the soluble, often denatured and reduced protein must be refolded. Several technically feasible alternatives to conduct IBs solubilization and on-column refolding have been proposed in recent years. However, rarely these on-column refolding alternatives have been evaluated from an economical point of view, questioning the feasibility of their implementation at a preparative scale. The presented study assesses the economic performance of four distinct process alternatives that include pH induced IBs solubilization and protein refolding (pH_IndSR); IBs solubilization using urea, dithiothreitol (DTT), and alkaline pH followed by batch size-exclusion protein refolding; inclusion bodies (IBs) solubilization using urea, DTT, and alkaline pH followed by simulated moving bed (SMB) size-exclusion protein refolding, and IBs solubilization using urea, DTT and alkaline pH followed by batch dilution protein refolding. The economic performance was judged on the basis of the direct fixed capital, and the production cost per unit of product (P(C)). This work shows that (1) pH_IndSR system is a relatively economical process, because of the low IBs solubilization cost; (2) substituting β-mercaptoethanol for dithiothreithol is an attractive alternative, as it significantly decreases the product cost contribution from the IBs solubilization; and (3) protein refolding by size-exclusion chromatography becomes economically attractive by changing the mode of operation of the chromatographic reactor from batch to continuous using SMB technology.     

Intracellular aggregation of polypeptides with expanded polyglutamine domain is stimulated by stress-activated kinase MEKK1

Abnormal proteins, which escape chaperone-mediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH(2)-terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulates several stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.     

The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А<Subscript>1</Subscript> from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> on the Structure and Properties of Inclusion Bodies

The effect of the concentration of an inducer (IPTG) and the time of induction at 37°С on the heterologous synthesis of the mature membrane protein phospholipase А₁ (PldA) from Yersinia pseudotuberculosis in the form of inclusion bodies (IBs) and on the physicochemical and structural characteristics of IBs has been studied. The sizes, shape, stability (solubility in urea and detergents, resistance against proteolysis), the secondary structure of the protein of IBs, and the presence of amyloid structures have been determined by electron microscopy, dynamic light scattering, and optical spectroscopy. It was found that IBs have a shape close to spherical and a rough surface and are cleaved by proteinase K. The protein contained in IBs has an ordered secondary structure with a high content of β-structure. As the inducer concentration and the time of expression increase, the conformation of the recombinant protein in IBs undergoes changes, as indicated by an increase in the stability of IBs and a decrease in the enzymatic activity of the protein. When IBs are dissolved in 0.06% SDS and 5 M urea, the recombinant protein retains the secondary structure in a partially modified form, and the addition of a zwitterionic detergent at a micellar concentration does not transform the protein conformation into the native one.     

Structures of l-fuculose-1-phosphate aldolase mutants outlining motions during catalysis

The crystal structures of l-fuculose-1-phosphate aldolase (FucA) with and without a ligated analogue of dihydroxyacetone phosphate (DHAP) and of a number of active center mutants have resulted in a model of the catalytic mechanism. This model has now been confirmed by structural analyses of further mutations at the zinc coordination sphere and at the phosphate site. In addition, these mutants have revealed new aspects of the catalysis: the hydroxyl group of Tyr113' (from a neighboring subunit), which sits just outside the zinc coordination sphere, steers DHAP towards a productive binding mode at the zinc ion; Glu73 contacts zinc in between the two ligand positions intended for the DHAP oxygen atoms and thus avoids blocking of these positions by a tetrahedrally coordinated hydroxy ion; the FucA polypeptide does not assume its minimum energy state but oscillates between two states of elevated energy as demonstrated by a mutant in a minimum energy state. The back and forth motion involves a mobile loop connecting the phosphate site with intersubunit motions and thus with the Brownian motion of the solvent. The phosphate group is bound strongly at a given distance to the zinc ion, which prevents the formation of too tight a DHAP:zinc complex. This observation explains our failure to find mutants that accept phosphate-free substitutes for DHAP. The FucA zinc coordination sphere is compared with that of carbonic anhydrase.     

Studies on the expression of recombinant fuculose-1-phosphate aldolase in E. coli

Fuculose-1-phosphate aldolase (Fuc-1-PA) is a dihydroxyacetone phosphate (DHAP) dependent aldolase with potential application in chiral synthesis. The influence of the growth medium on the expression of the enzyme in Escherichia coli has been studied. Complex LB medium, a defined medium (MD) and a semi-complex medium (MSC) have been compared in order to maximize aldolase production. The defined medium produced highest expression levels (700 activity units (AU)/g of dry cell weight (DCW)). The optimal induced isopropyl-β-thiogalactopyranoside (IPTG) concentration of 100 μM produces in the MD medium of 41 μmol/g dry cell weight of enzyme.     

Study of certain carbohydrate metabolism enzymes in an active oxytetracycline producer strain and in an inactive mutant in relation to antibiotic biosynthesis]

The activity of the glycolysis enzymes, i.e. aldolase and pyruvate decarboxylase and the enzymes of the pentose cycle, i.e. transketolase were investigated in the process of cultivation of an active strain and inactive mutant of Act. rimosus under conditions favourable for oxytetracycline biosynthesis on starch medium and under unfavourable conditions on glucose medium. It was shown that the aldolase and transketolase activity in the inactive mutant was higher on the starch medium as compared to the active strain, while the activity of pyruvate dekarboxylase was lower. The above difference between the both strains was preserved on the glucose medium and the activity of aldolase and transketolase in both strains increased, while the activity of pyruvate dekarboxylase remained at the same level.     

Kinetic mechanism of fuculose-1-phosphate aldolase from the hyperthermophilic archaeon Methanococcus jannaschii

Fuculose-1-phosphate aldolase (FucA) is a useful biocatalyst with potential applications in chiral synthesis. In this study, the overall kinetic mechanism of FucA from the archaeon Methanococcus jannaschii was studied. The K(m) values of dihydroxyacetone phosphate (DHAP) and dl-glyceraldehyde were 0.09 and 0.74 mM, respectively. Dead-end inhibition by trimethyl phosphonoacetate and dl-threose were competitive and uncompetitive with respect to DHAP and dl-glyceraldehyde. Inhibition patterns obtained using reaction products were noncompetitive vs. DHAP and competitive vs. dl-glyceraldehyde. The equilibrium constant was 8.309×10(-3) M as assessed by varying the [DHAP]/[product] ratio at a fixed dl-glyceraldehyde concentration and by measuring the change in DHAP concentration after equilibrium was reached. This constant is consistent with the K(eq) value obtained from (13)C NMR (15.625×10(-3) M). The resultant inhibition kinetics may suggest the insights of kinetic mechanism of the FucA catalyzed reaction.     

A simple feedback control of Escherichia coli growth for recombinant aldolase production in fed-batch mode

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.     

Influence of secondary reactions on the synthetic efficiency of DHAP-aldolases

The effect of secondary reactions on DHAP-dependent aldolase stereoselective synthesis yields is reported. The fuculose-1-phosphate aldolase catalyzed synthesis between DHAP and Cbz-S-alaninal has been chosen as case study. It has been demonstrated that DHAP is not only chemically degraded in the reaction medium, but also enzymatically. The last reaction has been shown to take place when type II aldolases are used as biocatalysts. In order to minimize the effect of non-desired reactions, temperature reduction has been shown to be favorable, and operation at 4 degrees C has been chosen as appropriate. On the other hand, the fed-batch addition of DHAP also increased the synthesis yields and, combined with low temperature, led to almost quantitative conversion.     

功能性包涵体的研究进展

利用原核系统表达外源重组蛋白的一个特点是表达蛋白多以包涵体形式存在。在过去人们一直认为包涵体是错误折叠、无生物活性的蛋白,需要经过变复性的过程重新获得有生物活性、可溶的蛋白,因而变复性条件的摸索迄今仍然是该领域的难点。但近几年的研究表明包涵体并非都是无生物活性的,功能性包涵体(或者称为非传统意义包涵体) 概念的提出是该领域的一个重大研究进展。由于功能性包涵体本身具有生物活性,可在非变性条件下提取,目前已经在生命科学的基础研究、生物制药、生物材料、生物催化等领域展现出良好的应用前景。重点从功能性包涵体的定义、形成机理、提取条件等近期研究进展进行综述,以期为原核细胞表达和工业生产重组蛋白药物提供新的思路。     

Reduction of N-terminal methionylation while increasing titer by lowering metabolic and protein production rates in E. coli auto-induced fed-batch fermentation

A standard fed-batch fermentation process using 1 mM isopropyl-β-D: -thiogalactopyranoside (IPTG) induction at 37 °C in complex batch and feed media had been developed for manufacturing of a therapeutic protein (TP) expressed in inclusion bodies (IBs) by E. coli BL21 (DE3) driven by T7 promoter. Six unauthentic TP N-terminal variants were identified, of which methionylated TP (Met-TP) ratio was predominant. We hypothesized that lowering metabolic and protein production rates would reduce the Met-TP ratio while improving TP titer. The standard process was surprisingly auto-induced without added IPTG due to galactose in the complex media. Without changing either the clone or the batch medium, a new process was developed using lower feed rates and auto-induction at 29 °C after glucose depletion while increasing induction duration. In comparison to the standard process, the new process reduced the unauthentic Met-TP ratio from 23.6 to 9.6 %, increased the TP titer by 85 %, and the specific production yield from 210 to 330 mg TP per gram of dry cell weight. Furthermore, the TP recovery yield in the purified IBs was improved by ~20 %. Adding together, ~105 % more TP recovered in the purified IBs from per liter of fermentation broth for the new process than the standard process. The basic principles of lowering metabolic and production rates should be applicable to other recombinant protein production in IBs by fed-batch fermentations.     

Packaging protein drugs as bacterial inclusion bodies for therapeutic applications

ABSTRACT: A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.     

Kinetic modelling of aldolase-catalyzed addition between dihydroxyacetone phosphate and (S)-alaninal

This paper is focused on the development of a kinetic model for an aldolase-catalyzed reaction. The aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-benzyloxycarbonyl-alaninal ((S)-Cbz-alaninal) catalyzed by the four DHAP-dependent aldolases is a promising way for the synthesis of four complementary diastereoisomers with potential biological activity. The reaction catalyzed by fuculose-1-phosphate aldolase (FucA) conducts to a synthesis product with a 100% diastereomeric excess. A kinetic model has been proposed including both the synthesis and a parallel non-desired secondary reaction. The model involved an ordered two-substrate mechanism for the synthesis and non-competitive inhibition by (S)-Cbz-alaninal and competitive inhibition by methylglyoxal byproduct in both reactions. The values of the model kinetic parameters were determined and the model validated in batch and fed-batch synthesis reactions. The obtained model could be extended to explain the behavior of other class II DHAP-dependent aldolases and exploited in simulation for reactor design purposes.     

Efficient solubilization of inclusion bodies

The overexpression of recombinant proteins in Escherichia coli leads in most cases to their accumulation in the form of insoluble aggregates referred to as inclusion bodies (IBs). To obtain an active product, the IBs must be solubilized and thereafter the soluble monomeric protein needs to be refolded. In this work we studied the solubilization behavior of a model-protein expressed as IBs at high protein concentrations, using a statistically designed experiment to determine which of the process parameters, or their interaction, have the greatest impact on the amount of soluble protein and the fraction of soluble monomer. The experimental methodology employed pointed out an optimum balance between maximum protein solubility and minimum fraction of soluble aggregates. The optimized conditions solubilized the IBs without the formation of insoluble aggregates; moreover, the fraction of soluble monomer was approximately 75% while the fraction of soluble aggregates was approximately 5%. Overall this approach guarantees a better use of the solubilization reagents, which brings an economical and technical benefit, at both large and lab scale and may be broadly applicable for the production of recombinant proteins.     

Performance of an immobilized fuculose-1-phosphate aldolase for stereoselective synthesis

A derivative of fuculose-1-phosphate aldolase, immobilized with high loading on glyoxal–agarose gels, has been characterized and evaluated as a biocatalyst for an aldol addition reaction. The reaction of the solid biocatalyst was diffusion-controlled for conversion of its natural substrate. Nevertheless, when catalyzing the synthesis of a biologically active aminopolyol, the lower reaction rate with non-natural substrates led to a process controlled by the intrinsic enzyme kinetics. The resulting biocatalyst has high synthetic specific activity and has been successfully used in batch synthesis reactions with high conversion. In addition, the immobilized aldolase has been employed in fed-batch synthesis, increasing the selectivity of the reaction and obtaining high conversion (88%).