Bacterial overexpression and purification of soluble recombinant human serum albumin using maltose-binding protein and protein disulphide isomerase.

Human serum albumin (HSA), the most abundant serum protein in healthy humans, plays important roles in many physiological processes and has wide clinical and research applications. Despite several efforts to obtain recombinant HSA (rHSA) from bacterial and eukaryotic expression systems, a low-cost and high-yield method for rHSA production is not available. The large molecular weight and high disulphide content hamper the expression and production of rHSA using bacterial hosts. Hence, a strategy that uses a fusion technique and engineered Escherichia coli strains was employed to improve the expression of soluble rHSA in the bacterial cytoplasm. The solubilities of the b'a' domain of human protein disulphide isomerase (PDIb'a')- and maltose-binding protein (MBP)-tagged rHSA expressed in Origami 2 at 18 °C were notably increased by up to 90.1% and 96%, respectively. A simple and efficient protocol for rHSA purification was established and approximately 9.46 mg rHSA was successfully obtained from a 500-mL culture at 97% purity. However, rHSA was mostly obtained in soluble oligomeric form. By introducing a simple refolding and size-exclusion chromatography step, monomeric rHSA was obtained at 34% yield. Native polyacrylamide gel electrophoresis confirmed the similarity in the molecular weights between E. coli-derived monomeric rHSA and commercial monomeric HSA.

Rational design of specific ligands for human serum albumin separation and applications.

Human serum albumin is widely used in clinical practice, and the development of new ligands with high affinity is beneficial to improve its separation efficiency. The Site II of human serum albumin is an active binding site of various molecules such as l-tryptophan, which was studied with molecular simulation to obtain insights for the design of new ligands. The results showed that the carboxyl and indolyl groups of l-tryptophan were critical for the binding on Site II. Seven ligands containing carboxyl groups and indolyl groups were designed, and molecular simulation showed that indole-3-pentanoic acid was the best ligand. A new ligand combined indole-3-acetic acid and cysteine was designed for easier resin preparation, and molecular simulation also indicated that the new ligand bound strongly to Site II. Resins with the new ligand designed was prepared and static adsorption experiments indicated that the new resin had high adsorption capacity of human serum albumin and strong salt tolerance. Finally, recombinant human serum albumin was separated from yeast broth with high purity of 90.4% and recovery of 94.2%, which indicated that the new resin had good adsorption selectivity and strong potential for applications.

Comparison of accompanying proteins in different therapeutic human serum albumin preparations.

Pharmaceutical human serum albumin products are manufactured from donated human plasma and may contain up to 5% accompanying non-albumin proteins. It has been reported that albumin preparations manufactured by different pharmaceutical companies differed in the degree of posttranslational modifications, the redox state as well as antioxidant properties of albumin, whereas the composition of the accompanying proteins has never been comparatively analyzed. In this study, a non-targeted mass spectrometric approach was used for label-free quantification and comparison of different pharmaceutical albumin preparations. Haptoglobin and a few other proteins accounted for approximately 80% of the accompanying proteins in all products tested. Low abundance proteins were enriched by means of a combinatorial peptide ligand library (ProteoMiner, Bio-Rad). Significant differences between the amounts of several mainly low abundance proteins, such as complement factors, were observed indicating differences in the manufacturing processes of the pharmaceutical companies. The removal of the stabilizers octanoate and N-acetyltryptophan from albumin solutions using the charcoal-based Hepalbin adsorbent simultaneously reduced the accompanying proteins. For therapy evaluation of albumin preparations, the variable composition of the accompanying proteins in different albumin products should be taken into account in addition to the known heterogeneity of the albumin protein itself.

A Near-infrared Turn-on Fluorescent Sensor for Sensitive and Specific Detection of Albumin from Urine Samples.

A readily synthesizable fluorescent probe DMAT-π-CAP was evaluated for sensitive and selective detection of human serum albumin (HSA). DMAT-π-CAP showed selective turn-on fluorescence at 730 nm in the presence of HSA with more than 720-fold enhancement in emission intensity ([DMAT-π-CAP] = 10 µM), and rapid detection of HSA was accomplished in 3 seconds. The fluorescence intensity of DMAT-π-CAP was shown to increase in HSA concentration-dependent manner (Kd = 15.4 ± 3.3 µM), and the limit of detection of DMAT-π-CAP was determined to be 10.9 nM (0.72 mg/L). The 1:1 stoichiometry between DMAT-π-CAP and HSA was determined, and the displacement assay revealed that DMAT-π-CAP competes with hemin for the unique binding site, which rarely accommodates drugs and endogenous compounds. Based on the HSA-selective turn-on NIR fluorescence property as well as the unique binding site, DMAT-π-CAP was anticipated to serve as a fluorescence sensor for quantitative detection of the HSA level in biological samples with minimized background interference. Thus, urine samples were directly analyzed by DMAT-π-CAP to assess albumin levels, and the results were comparable to those obtained from immunoassay. The similar sensitivity and specificity to the immunoassay along with the simple, cost-effective, and fast detection of HSA warrants practical application of the NIR fluorescent albumin sensor, DMAT-π-CAP, in the analysis of albumin levels in various biological environments.

Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

Predilution On-Line Hemodiafiltration Improves Albumin Redox in Maintenance Hemodialysis Patients.

BACKGROUND/AIM: In this study, we compared the dialysis efficiency, oxidative stress, and nutritional conditions between predilution on-line hemodiafiltration (pre-OL-HDF) and conventional hemodialysis (HD) using a super-flux dialyzer (CHD). METHOD: This was a crossover study of 38 maintenance HD patients. All patients were treated with CHD for the first 4 months (1st CHD period), then were switched to pre-OL-HDF for 4 months (pre-OL-HDF period), and were returned to CHD for the next 4 months (2nd CHD period). RESULTS: We found no significant difference in the removal ratio of small uremic substances or the indices of inflammation or nutritional states between the pre-OL-HDF and CHD periods. However, we found higher removal of ß2 micro-globulin in the pre-OL-HDF period, and the human mercapto-albumin (Alb)/human serum Alb ratio was significantly higher in the pre-OL-HDF period. CONCLUSION: Treatment with pre-OL-HDF enabled enhanced removal of middle molecule uremic toxins and better Alb redox than did CHD.

A novel purification procedure for recombinant human serum albumin expressed in Pichia pastoris.

Plasma-derived human serum albumin (pHSA) has important applications in many clinical indications, including blood loss, serious burn, or hemorrhagic shock. The limited supply and potential infectious pathogen contamination of pHSA have stimulated the development of recombinant human serum albumin (rHSA). However, rHSA often entraps endogenous or exogenous impurities, including color pigments and polysaccharides. Therefore, the purification of rHSA to high purity remains the bottleneck in large-scale production of rHSA. Herein we report a novel two-step purification protocol for rHSA generated from Pichia pastoris. In the first step, rHSA was partially unfolded to expose impurities entrapped into rHSA and was then removed. In the second step, rHSA was crystallized to further remove impurities. Through this procedure, the pigment content (A350/A280) and polysaccharides content of rHSA were reduced to 0.0141 and 0.253 µg/mg, respectively, which were comparable to pHSA. In addition, the secondary structure and bioactivity of purified rHSA are preserved. The purification procedure developed in this study is a simple, short and low cost method to produce active rHSA, or rHSA-fusion proteins, to high purity, and also suitable for the purification of other disulfide-rich proteins.

High level expression and purification of recombinant human serum albumin in Pichia pastoris.

Human serum albumin (HSA) has been extensively used in a series of clinical care settings for nearly seven decades. However, the broad application of this protein is seriously limited by its short supply. In this work, the codon sequence of HSA was cloned under the control of the alcohol oxidase 1 promoter (AOX1) and expressed as a secretory protein in Pichia pastoris. A recombinant strain displaying the highest HSA yield was selected by screening for resistance to the highest concentration of antibiotic G418. After optimizing the induction conditions and additional supplements, the highest yield of HSA reached 1.6 g/L in a shake flask. Performing high density fermentation further improved the highest yield to 8.86 g/L in a fermenter after 96 h of methanol induction. This result is more promising than the previous reports of industrial applications, which reported the highest yield as 92.29 mg/L/h, considering that the space-time yield of rHSA was doubled. In addition, the desired protein was purified by filtration and Cibacron Blue affinity chromatography, which yielded a 58% recovery of a product that had over a 96% purity. This study reveals that Pichia pastoris is an excellent system for recombinant human serum albumin expression due to its outstanding expression capacity. In addition, the high efficiency level of rHSA production lays a solid foundation for its use in industrial production.

Polymeric ionic liquid-assembled graphene-immobilized silica composite for selective isolation of human serum albumin from human whole blood.

Polymeric ionic liquids (PILs) with 1-vinyl-3-ethylimidazolium cations and two different anions of Br- and PF6- were assembled onto the surface of graphene (G) nanosheets. The derived two composites, i.e., PIL(Br)-G and PIL(PF6)-G, were further efficiently immobilized onto the surface of silica nanoparticles via self-assembly technique. The obtained two PIL-G/SiO2 nanocomposites exhibited diverse adsorption performances toward proteins through adjusting the anions of PILs. Electrostatic attractions between proteins and the nanocomposites dominated protein adsorption, while the presence of PF6- anions weakened electrostatic interactions and deteriorated the selective adsorption of target protein, i.e., bovine serum albumin (BSA) in this case. Specifically, PIL(Br)-G/SiO2 nanocomposite displayed high selectivity toward BSA and a high adsorption efficiency of ca. 98% was achieved for 100 mg L-1 BSA in a Britton-Robinson (B-R) buffer at pH 5. HPLC analysis demonstrated the selectivity of PIL(Br)-G/SiO2 nanocomposite toward BSA in the presence of abundant hemoglobin and cytochrome c. The practical applicability was verified by performing selective isolation of human serum albumin (HSA) from human whole blood. Graphical abstract Selective isolation of human serum albumin from blood by polymeric ionic liquid assembled graphene immobilized silica nanocomposite with tunable anions.

Simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis.

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.

Human albumin purification: a modified and concise method.

BACKGROUND: Among different proteins of blood, albumin is considered a unique protein due to having special properties. Now, various protocols are used for the albumin purification worldwide, each of them has its own advantages and disadvantages. Meanwhile, a common method which is often used for the production of albumin is a combination of Cohn along with different types of chromatography. The aim of the present study was to create a concise and cost-effective albumin purification method by employing a conventional method with some modifications. METHODS: In this research, the albumin was purified from human serum using chilled ethanol, followed by chromatographic methods. The purity of harvested albumin was evaluated by cellulose acetate membrane electrophoresis (CAME) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting (WB) analysis and thermostability were used for functional and stability measurement assessment, respectively. RESULTS: SDS-PAGE and CAME showed that the purity of purified human albumin was about 99%. Purified human albumin showed a single band with a molecular weight of 66 kDa. The results were validated by WB analysis .Also, the thermostability of purified albumin was same as the commercial albumin. CONCLUSION: This method can be a robust technique for purification of albumin in order to use clinical and research approaches.

Molecularly Imprinted Nanogels Acquire Stealth In†Situ by Cloaking Themselves with Native Dysopsonic Proteins.

Protein corona formation was regulated on the surface in vivo by molecular imprinting to enable polymeric nanogels to acquire stealth upon intravenous administration. Albumin, the most abundant protein in blood, was selected as a distinct protein component of protein corona for preparing molecularly imprinted nanogels (MIP-NGs) to form an albumin-rich protein corona. Intravital fluorescence resonance energy transfer imaging of rhodamine-labeled albumin and fluorescein-conjugated MIP-NGs showed that albumin was captured by MIP-NGs immediately after injection, forming an albumin-rich protein corona. MIP-NGs circulated in the blood longer than those of non-albumin-imprinted nanogels, with almost no retention in liver tissue. MIP-NGs also passively accumulated in tumor tissue. These data suggest that this strategy, based on regulation of the protein corona in vivo, may significantly influence the development of drug nanocarriers for cancer therapy.

Sample Displacement Batch Chromatography of Proteins.

In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.

Synthetic Ligand Affinity Chromatography Purification of Human Serum Albumin and Related Fusion Proteins.

Synthetic ligand affinity adsorbents offer an efficient means for purification of biopharmaceuticals. Single-isomer textile dye C.I. Reactive Blue and newer ligands developed by rational design and screening of chemical combinatorial libraries based on a triazine scaffold are routinely used for the capture and purification of these proteins from engineered recombinant expression systems. Here, we describe methods for the purification of recombinant human serum albumin and related fusion proteins using synthetic ligand affinity adsorbents.

An Orthogonal Fusion Tag for Efficient Protein Purification.

In this chapter, we present an efficient method for stringent protein purification facilitated by a dual affinity tag referred to as ABDz1, which is based on a 5 kDa albumin-binding domain from Streptococcal Protein G. The small fusion tag enables an orthogonal affinity purification approach based on two successive and highly specific affinity purification steps. This approach is enabled by native binding of ABDz1 to human serum albumin and engineered binding to Staphylococcal Protein A, respectively. The ABDz1-tag can be fused to either terminus of a protein of interest and the purification steps can be carried out using standard laboratory equipment.

Prognostic significance of pre-treatment ALBI grade in advanced non-small cell lung cancer receiving immune checkpoint therapy.

The liver is an essential organ for regulating innate and acquired immunity. We hypothesized that the pre-treatment hepatic function affects the clinical outcome of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC). We analyzed 140 patients with NSCLC who received ICIs. We investigated the association between pre-treatment liver function, assessed using the albumin-bilirubin (ALBI) grade, and clinical outcomes in univariate, multivariate, and propensity score matching analyses. Patients were divided into four grades according to pre-treatment liver function. Eighty-eight patients had good hepatic reserve (ALBI grade 1 or 2a), whereas 52 patients had poor hepatic reserve (ALBI grade 2b or 3). In the univariate Kaplan-Meier analysis, the ALBI grade 1, 2a group had a significantly prolonged progression-free survival (PFS, 5.3 versus 2.5 months, p = 0.0019) and overall survival (OS, 19.6 vs. 6.2 months, p = 0.0002). These results were consistent, regardless of whether the analysis was performed in patients with a performance status of 0 or 1 at pre-treatment (N = 124) or in those selected using propensity score matching (N = 76). In the multivariate analysis, pre-treatment ALBI grade was an independent prognostic factor for both PFS (hazard ratio [HR] 0.57, 95% confidence interval [95% CI] 0.38-0.86, p = 0.007) and OS (HR 0.45, 95% CI 0.29-0.72, p = 0.001). Our results suggest that pre-treatment hepatic function assessed by ALBI grade could be an essential biomarker for predicting the efficacy of treatment with ICIs in NSCLC.

Blocking the Nanopores in a Layer of Nonconductive Nanoparticles: Dominant Effects Therein and Challenges for Electrochemical Impedimetric Biosensing.

Blockage of a nanopore by an analyte molecule has emerged as a promising concept for electrochemical biosensing. Nanoporous structures can be formed on the electrode surface simply by packing spherical nanoparticles in a dense planar arrangement. Modification of the nanoparticles with human serum albumin (HSA) and its interaction with the corresponding antibody (anti-HSA) can induce nanopore-blockage which significantly hinders permeation of the redox probe ([Fe(CN6)]4-/3-). Interfaces of different parameters were studied using Electrochemical Impedance Spectroscopy (EIS), and counterintuitively, the influence of charge of the nanoparticles and other immobilized entities played a substantial role in the measurement. Our study reveals dominant effects including the presence of mixed output signal and resolves corresponding EIS biosensing-related challenges. Consequently, blocking the nanopores was introduced as an efficient technique which enables the application of EIS-based biosensing to real-world analytical issues.

A dual-step immobilization/imprinting approach to prepare magnetic molecular imprinted polymers for selective removal of human serum albumin.

One viable solution to improve the conformational stability of template proteins is to use multiple, weaker modes of action to immobilize proteins on the surfaces of a solid support. Herein, we introduce a novel surface imprinting technique for human serum albumin (HSA) by a dual immobilization/imprinting strategy. Specifically, HSA was first conjugated to the surfaces of magnetic Fe3O4 nanoparticles through a reversible aldmine condensation reaction. Dopamine (DA) was then used to imprint the protein template via an auto-polymerization reaction in biocompatible aqueous media. The resultant magnetic molecular imprinted polymers (MMIPs) possess high adsorption capacity (70.2 mg g-1), superior selectivity (IF = 4.54), and rapid capturing kinetics to HSA (within 20 min). We successfully demonstrate the practical applicability of MMIPs to the selective removal of HSA from human serum sample. Our work offers a novel and robust solution to develop proteins imprinted materials with high binding capacity and selectivity. We anticipate such materials will find wide applications to protein detection or removal in diverse real-life clinical and biological samples.

A micro-solid phase extraction device to prepare a molecularly imprinted porous monolith in a facile mode for fast protein separation.

A molecularly imprinted polymeric monolith was synthesized in an aqueous environment in 15 min via UV-irradiation. The imprinted monolith was composed of hydroxyethyl methacrylate as monomer, dimethyl amino ethyl methacrylate as functional monomer, methylene bisacrylamide and piperazine diacrylamide as crosslinkers and human serum albumin as template molecule. The synthesis took place in a PDMS-based device (2.5 cm long) yielding a micro-solid phase extraction column (3 × 5 mm) with two built-in fingertight connectors for an infusion pump and fraction collector. The imprinted monolith displayed the characteristic features of a porous polymeric monolith, had dimethyl amino ethyl methacrylate and human serum albumin as functional groups within the monolith and showed high permeability (0.51 × 10-13 m2). 85% of the imprinted cavities were readily available for rebinding of human serum albumin with an imprinting factor of 1.3. In comparison to a non-imprinted monolith, molecular imprinting increased human serum albumin adsorption by > 30%. Imprinted monolith displayed selectivity for human serum albumin over other competing proteins (human transferrin, ovalbumin and carbonic anhydrase) with similar or different isoelectric points and size. Human serum albumin was adsorbed (in dynamic mode) with > 98% selectivity from diluted human plasma using the imprinted monolith device. Device to device reproducibility and reusability of the device for 5 cycles showcase the imprinted monolith micro-device efficiency.