Enhanced Protein Stability Research Articles

FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane.

Mutations in the skeletal muscle-specific CLC-1 chloride channel are associated with the human hereditary disease myotonia congenita. The molecular pathophysiology underlying some of the disease-causing mutations can be ascribed to defective human CLC-1 protein biosynthesis. CLC-1 protein folding is assisted by several molecular chaperones and co-chaperones, including FK506-binding protein 8 (FKBP8). FKBP8 is generally considered an endoplasmic reticulum- and mitochondrion-resident membrane protein, but is not thought to contribute to protein quality control at the cell surface. Herein, we aim to test the hypothesis that FKBP8 may regulate CLC-1 protein at the plasma membrane. Surface biotinylation and subcellular fractionation analyses reveal that a portion of FKBP8 is present at the plasma membrane, and that co-expression with CLC-1 enhances surface localization of FKBP8. Immunoblotting analyses of plasma membrane proteins purified from skeletal muscle further confirm surface localization of FKBP8. Importantly, FKBP8 promotes CLC-1 protein stability at the plasma membrane. Together, our data underscore the importance of FKBP8 in the peripheral quality control of CLC-1 channel.

Enhancing Protein Stability with Genetically Encoded Noncanonical Amino Acids.

The ability to add noncanonical amino acids to the genetic code may allow one to evolve proteins with new or enhanced properties using a larger set of building blocks. To this end, we have been able to select mutant proteins with enhanced thermal properties from a library of E. coli homoserine O-succinyltransferase ( metA) mutants containing randomly incorporated noncanonical amino acids. Here, we show that substitution of Phe 21 with ( p-benzoylphenyl)alanine (pBzF), increases the melting temperature of E. coli metA by 21 °C. This dramatic increase in thermal stability, arising from a single mutation, likely results from a covalent adduct between Cys 90 and the keto group of pBzF that stabilizes the dimeric form of the enzyme. These experiments show that an expanded genetic code can provide unique solutions to the evolution of proteins with enhanced properties.

Protein evolution speed depends on its stability and abundance and on chaperone concentrations

Proteins evolve at different rates. What drives the speed of protein sequence changes? Two main factors are a protein's folding stability and aggregation propensity. By combining the hydrophobic-polar (HP) model with the Zwanzig-Szabo-Bagchi rate theory, we find that: (i) Adaptation is strongly accelerated by selection pressure, explaining the broad variation from days to thousands of years over which organisms adapt to new environments. (ii) The proteins that adapt fastest are those that are not very stably folded, because their fitness landscapes are steepest. And because heating destabilizes folded proteins, we predict that cells should adapt faster when put into warmer rather than cooler environments. (iii) Increasing protein abundance slows down evolution (the substitution rate of the sequence) because a typical protein is not perfectly fit, so increasing its number of copies reduces the cell's fitness. (iv) However, chaperones can mitigate this abundance effect and accelerate evolution (also called evolutionary capacitance) by effectively enhancing protein stability. This model explains key observations about protein evolution rates.

Aggressive conditions during primary drying as a contemporary approach to optimise freeze-drying cycles of biopharmaceuticals

Freeze-drying is the method of choice to dry formulations with biopharmaceutical drugs, to enhance protein stability. This is usually done below the glass transition temperature of maximally freeze-concentrated solutions (Tg′), to avoid protein aggregation, preserve protein activity, and obtain pharmaceutically ‘elegant’ cakes. Unfortunately, this is a lengthy and energy-consuming process. However, it was recently shown that drying above Tg′ or even above the collapse temperature (Tc) is not necessarily detrimental for stability of biopharmaceuticals, and hence provides an attractive option for freeze-drying cycle optimisation. The goal of the present study was to optimise the freeze-drying cycle for a model IgG monoclonal antibody (20 mg/mL) in sucrose and sucrose/glycine formulations, by reducing primary drying time. To study the impact of shelf temperature (Ts) and chamber pressure on product temperature (Tp), one conventional and five aggressive cycles were tested. Aggressive conditions during primary drying were achieved by increasing Ts from −20 °C (conventional cycle) to 30 °C, with chamber pressure set to 0.1 mbar, 0.2 mbar or 0.3 mbar. These combinations of Ts and chamber pressure resulted in Tp well above Tg′, and in some cases, even above Tc, without causing macrocollapse. Other critical quality attributes of the products were also within the expected ranges, such as reconstitution time and residual water content. Physical stability was tested using size exclusion chromatography, dynamic light scattering, and micro-flow imaging. All of the lyophilised samples were exposed to stress and the intended storage conditions, with no impacts on the product seen. These data show that implementation of aggressive conditions for the investigated formulations is possible and can significantly contribute to the reduction of primary drying times by up to 54% (from 48 to 22 h) in comparison to conventional freeze-drying.

Abstract LB-188: POH1 knockdown induces cancer cell apoptosis via p53 and Bim

Abstract The ubiquitin-proteasome system is implicated in cell apoptosis that is frequently dysregulated in human cancers. POH1/rpn11/PSMD14, as a part of the 19S proteasomal subunit, contributes to the progression of malignancy, but its role in apoptosis remains unclear. Here, we showed that POH1 expression was increased and associated with poor outcomes in three independent cohorts of patients with hepatocellular carcinoma (HCC), esophageal cancer (EC), and colorectal cancer (CRC). The knockdown of POH1 significantly inhibited tumor cell proliferation and induced apoptosis mediated by the mitochondrial pathway in vitro. Intratumoral injection of POH1 small interfering RNA (siRNA) significantly reduced the progression of tumor growth and induced apoptosis in vivo. Furthermore, p53 or Bim siRNA markedly attenuated the apoptosis induced by POH1 depletion. POH1 depletion resulted in cell apoptosis by increasing the stability of p53 and Bim and inhibiting their ubiquitination. Overall, POH1 knockdown induced cell apoptosis through increased expression of p53 and Bim via enhanced protein stability and attenuated degradation. Thus, POH1 may serve as a potential prognostic marker and therapeutic target in human cancers. Citation Format: Shi-Lu Chen. POH1 knockdown induces cancer cell apoptosis via p53 and Bim [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-188.

Abstract 4448: Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer

Abstract The Speckle type Poz Protein (SPOP) is a tumor suppressor that is often mutated in prostate cancer. Mutation of SPOP is linked to genomic instability and it plays a critical role in prostate cancer initiation. SPOP mutations are found to be in the heterozygous state in patients. The relevance of these mutations on its protein expression has not been systematically investigated. Here we report that mutations in the center MATH domain of SPOP lead to enhanced protein stability in prostate cancer. Among the mutations, F102C, S119N, F125V and WI31G showed increased protein stability. Further we show that these mutations in the MATH domain increase half-life of SPOP protein. Interestingly, the mutant SPOPs enhanced the endogenous wild-type SPOP level. We further explored the structural insights of these SPOP mutants. We present a computational model to predict how SPOP mutations potentially lead to conformational changes within the protein, promoting its stability. Finally, immunohistochemistry in clinical samples of prostate cancer augment our findings. Together, we highlight a subset of clinically relevant SPOP mutations that have impact on endogenous SPOP protein stability, allowing to further understand its role in prostate cancer initiation and evaluating of potentially altered therapeutic response in patients harboring these mutations. Citation Format: Joshua Fried, Vinayak Khattar, Jinlu Ma, Qinghua Zeng, Wei Zhang, Bo Xu. Mutations in the center MATH domain of Speckle Type BTB/POZ Protein lead to enhanced proteinstability in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4448.

Single-Molecule Spectroscopy Study of Crowding-Induced Protein Spontaneous Denature and Crowding-Perturbed Unfolding-Folding Conformational Fluctuation Dynamics.

The effects of molecular crowding on protein folding-unfolding processes are of importance for understanding protein function and structure dynamics in living cells. The enhancement of protein stability as a result of reduced entropic effect in the presence of molecular crowding is well understood both experimentally and theoretically. However, because of the complexity and interplay between various interactions existing in an equally favored environment of protein folding and unfolding conformational dynamics, such a simple reduced entropic enhancement model does not suffice to describe protein folding conformational dynamics under a protein crowding condition. In this paper, we report our observation on that single protein molecules spontaneously denature into unfolded proteins and folding-unfolding fluctuations in solution of crowding reagent Ficoll 70. We have identified that such protein dynamics involves a combined mechanism of polymer-polymer interaction, entropic effects, and protein solvation dynamics. We characterize the protein folding-unfolding dynamics by using single-molecule spectroscopy to obtain detailed molecular dynamic scale information on the protein folding-unfolding conformational fluctuation dynamics. Our findings suggest that the complex unfolding dynamic processes are spontaneous denature of single protein molecules induced by molecular crowding effect which has been elusive for analysis in ensemble-averaged measurements. Furthermore, the energy needed for the spontaneous unfolding is at the biological accessible force fluctuation level, which suggests a strong implication of significant human health relevance and importance. The new knowledge of the inhomogeneous protein unfolding processes can serve as a step forward to a mechanistic understanding of human diseases associated with molecular crowding, protein aggregates, fibril formation, as well as gene translational regulation processes typically under a molecular crowded local environment.

One-Pot, One-Step Production of Dietary Nucleotides by Magnetic Biocatalysts

The enzymatic synthesis of nucleotides offers several advantages over traditional multistep chemical methods, such as stereoselectivity, regioselectivity, enantioselectivity, simple downstream processing, and the use of mild reaction conditions. However, in order to scale up these bioprocesses, several drawbacks, such as the low enzyme stability and recycling, must be considered. Enzyme immobilization may overcome these cost-related problems by enhancing protein stability and facilitating the separation of products. In this regard, tetrameric hypoxanthine–guanine–xanthine phosphoribosyltransferase (HGXPRT) from Thermus thermophilus HB8 was covalently immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtHGXPRT). In this context, two different strategies were followed: (a) an enzyme immobilization through its N-terminus residues at pH 8.5 (derivatives MTtHGXPRT1-3); and (b) a multipoint covalent immobilization through the surface lysine residues at pH 10 (derivatives MTtHGXPRT4-5). The immobilized derivatives of MTtHGXPRT3 (activity 1581 international units per gram of support, IU/g; retained activity 29%) and MTtHGXPRT5 (activity 1108 IU/g; retained activity 23%) displayed the best wet biocatalyst activity, and retained activity values in the enzymatic synthesis of inosine-5′-monophosphate (IMP). In addition, the dependence of the activities and stabilities of both derivatives on pH and temperature was tested, as well as their reusability potential. Taking these results into account, MTtHGXPRT3 was chosen as the best biocatalyst (negligible loss of activity at 60 °C during 24 h; reusable up to seven cycles). Finally, as proof of concept, the enzymatic production of dietary nucleotides from high concentrations of low soluble bases was achieved.

Triptolide up-regulates metabotropic glutamate receptor 5 to inhibit microglia activation in the lipopolysaccharide-induced model of Parkinson’s disease

Metabotropic glutamate receptor (mGlu)5 regulates microglia activation, which contributes to inflammation. However, the role of mGlu5 in neuroinflammation associated with Parkinson’s disease (PD) remains unclear. Triptolide (T10) exerts potent immunosuppressive and anti-inflammatory effects and protects neurons by inhibiting microglia activation. In this study, we investigated the role of mGlu5 in the anti-inflammatory effect of T10 in a lipopolysaccharide (LPS)-induced PD model. In cultured BV2 cells and primary microglia, blocking mGlu5 activity or knocking down its expression abolished T10-inhibited release of proinflammatory cytokines induced by LPS. Moreover, T10 up-regulated mGlu5 expression decreased by LPS through enhancing mRNA expression and protein stability. T10 also reversed the reduction in mGlu5 membrane localization and modulated receptor-mediated mitogen-activated protein kinase activity induced by LPS. Pharmacological inhibition of signaling molecules increased nitric oxide level and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α, and interleukin (IL)-1β and -6 transcript levels that were downregulated by treatment with T10. Consistent with these in vitro findings, blocking mGlu5 attenuated the anti-inflammatory effects of T10 in an LPS-induced PD model and blocked the decreases in the number and morphology of ionized calcium binding adaptor molecule 1-positive microglia and LPS-induced iNOS protein expression caused by T10 treatment. Besides, mGlu5 mediated the effect of T10 on microglia-induced astrocyte activation in vitro and in vivo. The findings provide evidence for a novel mechanism by which mGlu5 regulates T10-inhibited microglia activation via modulating protein expression of the receptor and its intracellular signaling. The study might contribute to the biological effects of Chinese herbs as an approach for protecting against neurotoxicity in PD.

Pasteurella multocida PfhB2 toxin displays a novel unconventional cysteine protease fold

Pasteurella multocida causes respiratory tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multi‐domain toxin, called PfhB2, which contains a YopT‐like putative cysteine protease domain at its C‐terminus. PfhB2's YopT domain contains a well‐conserved Cys‐His‐Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. PfhB2 is implicated in virulence. Its FHA (filamentous hemagglutinin) and Fic (filamention induced by cAMP) domains have been shown to provide protective immunity in turkeys and to adenylylate Rho‐GTPases in vitro, respectively. In contrast, little is known about PfhB2's YopT‐like domain. Here, we show that PfhB2‐YopT does not display cysteine protease activity in a manner similar to Yersinia YopT or any other characterized YopT family members. We, therefore, opted to characterize PfhB2‐YopT structurally. Using a catalytically inactive C3733S mutant of PfhB2‐YopT that provides enhanced protein stability, we determined the crystal structure of PfhB2‐YopTC3733S solved by experimental phasing with Selenomethionine derivative crystals at 2.2Å resolution. Our structure shows that PfhB2‐YopT exists as four molecules arranged as an asymmetric unit consisting of two dimers. Importantly, the Cys from each molecule of the dimer coordinates with the His and Asp from the other molecule to complete the catalytic triad. Such an unconventional orientation of the catalytic triad has never been observed for any class of Cysteine proteases. Thus, our PfhB2‐YopT structure represents a novel cysteine protease fold, and provides critical insights for the function of Cys‐His‐Asp containing domains that are often associated with large, multi‐domain bacterial toxins.Support or Funding InformationThis work was supported by funding from 1) an Indiana CTSI (Clinical and Translational Sciences Institute) Project Development Team Award and 2) the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) under Award Number R01GM100092.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

POH1 Knockdown Induces Cancer Cell Apoptosis via p53 and Bim

The ubiquitin-proteasome system is implicated in cell apoptosis that is frequently dysregulated in human cancers. POH1/rpn11/PSMD14, as a part of the 19S proteasomal subunit, contributes to the progression of malignancy, but its role in apoptosis remains unclear. Here, we showed that POH1 expression was increased and associated with poor outcomes in three independent cohorts of patients with hepatocellular carcinoma (HCC), esophageal cancer (EC), and colorectal cancer (CRC). The knockdown of POH1 significantly inhibited tumor cell proliferation and induced apoptosis mediated by the mitochondrial pathway in vitro. Intratumoral injection of POH1 small interfering RNA (siRNA) significantly reduced the progression of tumor growth and induced apoptosis in vivo. Furthermore, p53 or Bim siRNA markedly attenuated the apoptosis induced by POH1 depletion. POH1 depletion resulted in cell apoptosis by increasing the stability of p53 and Bim and inhibiting their ubiquitination. Overall, POH1 knockdown induced cell apoptosis through increased expression of p53 and Bim via enhanced protein stability and attenuated degradation. Thus, POH1 may serve as a potential prognostic marker and therapeutic target in human cancers.

Polymer chain protects proteins

A new random heteropolymer with mixed hydrophilic and hydrophobic groups coats proteins to help them remain folded and active in environments that would normally unfold and inactivate them (Science 2018, DOI: 10.1126/science.aao0335). Random heteropolymers have been designed before to enhance the properties of small molecules, but not proteins. And researchers have previously enhanced protein stability by incorporating proteins in micelles or sol-gel networks or by combining proteins with polyelectrolytes and other polymers. But the new heteropolymer preserves native protein function and structure more faithfully in both aqueous and organic media, say Ting Xu of the University of California, Berkeley, and coworkers, who developed it. The heteropolymer could have bioremediation applications and allow researchers to make proteins without the help of cells. “This is outstanding work showing the ability of functionally diverse, random copolymers to act as chaperones and protein stabilizers for diverse applica...

YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys-His-Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5 Å resolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2 Å resolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c=74.7 Å for the native crystals and a=139.2, b = 139.2, c = 74.7 Å for the iodide-derivative crystals, are discussed.

BPTI folding revisited: switching a disulfide into methylene thioacetal reveals a previously hidden path.

Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein that is stabilized by three disulfide bonds at positions 5-55, 14-38 and 30-51. Widely studied for about 50 years, BPTI represents a folding model for many disulfide-rich proteins. In the study described below, we replaced the solvent exposed 14-38 disulfide bond with a methylene thioacetal bridge in an attempt to arrest the folding pathway of the protein at its two well-known intermediates, N' and N*. The modified protein was expected to be unable to undergo the rate-determining step in the widely accepted BPTI folding mechanism: the opening of the 14-38 disulfide bond followed by rearrangements that leads to the native state, N. Surprisingly, instead of halting BPTI folding at N' and N*, we uncovered a hidden pathway involving a direct reaction between the N* intermediate and the oxidizing reagent glutathione (GSSG) to form the disulfide-mixed intermediate N*-SG, which spontaneously folds into N. On the other hand, N' was unable to fold into N. In addition, we found that the methylene thioacetal bridge enhances BPTI stability while fully maintaining its structure and biological function. These findings suggest a general strategy for enhancing protein stability without compromising on function or structure, suggesting potential applications for future therapeutic protein production.

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

Improving protein stability is an important goal for basic research as well as for clinical and industrial applications but no commonly accepted and widely used strategy for efficient engineering is known. Beside random approaches like error prone PCR or physical techniques to stabilize proteins, e.g. by immobilization, in silico approaches are gaining more attention to apply target-oriented mutagenesis. In this review different algorithms for the prediction of beneficial mutation sites to enhance protein stability are summarized and the advantages and disadvantages of FoldX are highlighted. The question whether the prediction of mutation sites by the algorithm FoldX is more accurate than random based approaches is addressed.

Protein-solvent interaction.

Protein folding and assembly can be manipulated in in vitro systems by co-solvents at high concentrations. A number of co-solvents that enhance protein stability and assembly have been shown to be excluded from the protein surface. Such co-solvent exclusion has been demonstrated by dialysis experiments and shown to be correlated with their effects on protein stability and assembly.

Oxetane Grafts Installed Site‐Selectively on Native Disulfides to Enhance Protein Stability and Activity In Vivo

AbstractA four‐membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site‐selective bis‐alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb‐Aβ against the human Amyloid‐β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.

Oxetane Grafts Installed Site-Selectively on Native Disulfides to Enhance Protein Stability and Activity In Vivo.

A four‐membered oxygen ring (oxetane) can be readily grafted into native peptides and proteins through site‐selective bis‐alkylation of cysteine residues present as disulfides under mild and biocompatible conditions. The selective installation of the oxetane graft enhances stability and activity, as demonstrated for a range of biologically relevant cyclic peptides, including somatostatin, proteins, and antibodies, such as a Fab arm of the antibody Herceptin and a designed antibody DesAb‐Aβ against the human Amyloid‐β peptide. Oxetane grafting of the genetically detoxified diphtheria toxin CRM197 improves significantly the immunogenicity of this protein in mice, which illustrates the general utility of this strategy to modulate the stability and biological activity of therapeutic proteins containing disulfides in their structures.

Enhanced Protein Stability Using Room Temperature Ionic Liquid for Wearable Electrochemical Sweat-Based Biosensor

In this abstract we demonstrate the implementation of a hybrid aqueous/room temperature ionic liquid (RTIL) interface towards the development of a wearable biosensor for detection of inflammation through the use of a diagnostics protein biomarker. RTILs have low vapor pressure and a wide electrochemical window. RTILs are also known to stabilize proteins based on their physical properties such as hydrophobicity or hydrophilicity, viscosity and the chemical interaction of the RTIL with various functional groups of a protein. Typically, proteins are stable in RTILs with chaotropic cation and kosmotropic anion. Also, the interaction of anionic moiety with protein influences the protein stability as compared to the cationic moiety. We leverage the physicochemical properties of the RTIL, BMIM[BF4], along with its excellent electrochemical properties towards the development of a wearable electrochemical biosensor for the detection of IL-6 in human sweat. Firstly, the stability of α IL-6 antibody in RTIL was evaluated using optical techniques such as ATR-FTIR, dynamic light scattering (DLS) to determine any conformational changes or aggregation of the protein. ATR-FTIR temporal studies showed that the antibody was more stable in BMIM[BF4] (up to 96h) without any peak shifts as compared to PBS (up to 48h) and no aggregation in protein size was observed using DLS for up to 96h in BMIM[BF4]. Further, high zeta potential values for volumetric ratios ≥50% BMIM[BF4] in synthetic sweat(pH 2- 8) indicate that the protein does not aggregate in the presence of BMIM[BF4]. Once the enhanced stability of protein in BMIM[BF4] was confirmed, non-faradaic electrochemical impedance spectroscopy (EIS) was performed to assess the sensor performance metrics for detection of interleukin -6 (IL-6) in human sweat. The wearable sensor with metal-semiconductor electrode functionalized with IL-6 antibody in RTIL showed a limit of detection of 0.2pg/mL up to 24h, 2 pg/mL between 24- 48h, 5pg/mL between 48- 96h post-antibody functionalization. This LOD is below the physiological relevant range of 7-16 pg/mL. The linear dynamic range of the sensor obtained was 0.2- 200 pg/mL. This enhanced sensor performance is not only due to the protein stability maintained by the RTIL but also because of the electrochemical characteristics of RTIL that contributed to the enhanced electrical double layer post 96h immobilization. The physical, electrochemical along with protein stabilization properties of RTIL make it novel for electrochemical protein biosensing.

Addition of Pullulan to Trehalose Glasses Improves the Stability of β-Galactosidase at High Moisture Conditions

Incorporation of therapeutic proteins in a matrix of sugar glass is known to enhance protein stability, yet protection is often lost when exposed to high relative humidity (RH). We hypothesized that especially in these conditions the use of binary glasses of a polysaccharide and disaccharide might yield advantages for protein stability. Therefore, different amounts of the polysaccharide pullulan were introduced in freeze-dried trehalose glasses. In these homogeneous blends, the presence of pullulan above 50 weight % prevented crystallization of trehalose when exposed to high RH. Storage stability testing up to 4 weeks of the model protein β-galactosidase incorporated in pullulan/trehalose blends showed superior behavior of pure trehalose at 30°C/0% RH, while pullulan/trehalose blends yielded the best stability at 30°C/56% RH. In conclusion, binary glasses of pullulan and trehalose may provide excellent stability of proteins under storage conditions that may occur in practice, namely high temperature and high RH.