Medium optimization for high yield production of human serum albumin in Pichia pastoris and its efficient purification.

OBJECTIVE: To improve the yield of recombinant human serum albumin (HSA) in Pichia pastoris by medium optimization and establish the related purification scheme. RESULTS: A simplified version of the generally used buffered glycerol complex medium (BMGY), which contained yeast extract, glycerol and potassium salts, was found to be applicable. By decreasing the salt concentration of basal salt medium (BSM) to half of the original formula further, we achieved a high yield of 17.47 g/L HSA in the supernatant within a 192 h induction, which is the highest rHSA yield ever reported as far as we know. Accompanied with a three-step purification procedure which recovered two thirds of the desired protein at high purity, our work lays a solid foundation for large-scale industrial production of HSA. CONCLUSION: Medium optimization plays a significant role in improving the yield of desired protein, lowering the production cost and helping to explore the producing strain's character.

Transient expression of human serum albumin (HSA) in tobacco leaves.

Today, recombinant human proteins make up a considerable part of FDA-approved biotechnological drugs. The selection of proper expression platform for manufacturing recombinant protein is a vital factor in achieving the optimal yield and quality of a biopharmaceutical in a timely fashion. This experiment was aimed to compare the transient expression level of human serum albumin gene in different tobacco genotype. For this, the Agrobacterium tumefaciens strains LB4404 and GV3101 harboring pBI121-HSA binary vector were infiltered in leaves of three tobacco genotypes, including Nicotiana benthamiana and N. tabacum cv Xanthi and Samsun. The qRT-PCR, SDS-PAGE, western blotting and ELISA analysis were performed to evaluate the expression of HSA gene in transgenic plantlets. Our results illustrated that the expression level of rHSA in tobacco leaves was highly dependent on Agrobacterium strains, plant genotypes and harvesting time. The highest production of recombinant HSA protein was obtained in Samsun leaves infected with A. tumefaciens strain GV3101 after 3 days of infiltration.

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.

Antioxidant and reactive oxygen species scavenging properties of cellular albumin in HepG2 cells is mediated by the glutathione redox system.

This study was carried out to examine the role of intracellular albumin in the modulation of oxidative damage induced by glutathione modifiers in HepG2 cells. Also, the relationship of albumin synthesis with oxidative stress factors including antioxidants was studied. HepG2 cell culture was supplemented with glutathione modifiers; L-Buthionine-sulfoximine (BSO; 0.1 and 1.0 mM) or N-acetyl cysteine (NAC; 1 and 10 mM) and the cell viability and changes in reduced glutathione (GSH), oxidized glutathione (GSSG), reactive oxygen species (ROS), catalase, and superoxide dismutase were measured. Besides, albumin expression at protein and mRNA levels was determined in cells pretreated with BSO or NAC. Kinetic studies showed that albumin expression in HepG2 cells is correlated with GSH and GSSG levels. Changes in albumin expression at protein and mRNA levels reached their maximum (19% and 55%, respectively) in the cells 6 H after NAC treatments. A substantial decrease in intracellular albumin due to BSO (27%) was associated with a significant increase in the generation of cellular ROS (17%). In contrast, increased albumin synthesis (intracellular and secretory) was associated with inhibition in cellular ROS. Overall data may suggest that albumin expression in coordination with the glutathione redox system is part of the antioxidant defense mechanism in liver cells.

High-efficiency production of human serum albumin in the posterior silk glands of transgenic silkworms, Bombyx mori L.

Human serum albumin (HSA) is an important biological preparation with a variety of biological functions in clinical applications. In this study, the mRNA of a fusion transposase derived from the pESNT-PBase plasmid and a pBHSA plasmid containing the HSA gene under the control of a fibroin light chain (FL) promoter were co-injected into fertilized eggs. Fifty-six transgenic silkworm pedigrees expressing theexogenous recombinant HSA (rHSA) in the posterior silk glands (PSGs) with stable inheritance were successfully obtained. The SDS-PAGE and Western blot results confirmed that the rHSA was secreted into the transgenic silkworm cocoon, and the rHSA could be easily extracted with phosphate-buffered saline (PBS). In our research, the isolated highest amount rHSA constituted up to 29.1% of the total soluble protein of the cocoon shell, indicating that the transgenic silkworm produced an average of 17.4 µg/mg of rHSA in the cocoon shell. The production of soluble rHSA in the PSGs by means of generating transgenic silkworms is a novel approach, whereby a large amount of virus-free and functional HSA can be produced through the simple rearing of silkworms.

Revisiting Escherichia coli as microbial factory for enhanced production of human serum albumin.

BACKGROUND: Human serum albumin (HSA)-one of the most demanded therapeutic proteins with immense biotechnological applications-is a large multidomain protein containing 17 disulfide bonds. The current source of HSA is human blood plasma which is a limited and unsafe source. Thus, there exists an indispensable need to promote non-animal derived recombinant HSA (rHSA) production. Escherichia coli is one of the most convenient hosts which had contributed to the production of more than 30% of the FDA approved recombinant pharmaceuticals. It grows rapidly and reaches high cell density using inexpensive and simple subst-rates. E. coli derived recombinant products have more economic potential as fermentation processes are cheaper compared to the other expression hosts. The major bottleneck in exploiting E. coli as a host for a disulfide-rich multidomain protein is the formation of aggregates of overexpressed protein. The majority of the expressed HSA forms inclusion bodies (more than 90% of the total expressed rHSA) in the E. coli cytosol. Recovery of functional rHSA from inclusion bodies is not preferred because it is difficult to obtain a large multidomain disulfide bond rich protein like rHSA in its functional native form. Purification is tedious, time-consuming, laborious and expensive. Because of such limitations, the E. coli host system was neglected for rHSA production for the past few decades despite its numerous advantages. RESULTS: In the present work, we have exploited the capabilities of E. coli as a host for the enhanced functional production of rHSA (~ 60% of the total expressed rHSA in the soluble fraction). Parameters like intracellular environment, temperature, induction type, duration of induction, cell lysis conditions etc. which play an important role in enhancing the level of production of the desired protein in its native form in vivo have been optimized. We have studied the effect of assistance of different types of exogenously employed chaperone systems on the functional expression of rHSA in the E. coli host system. Different aspects of cell growth parameters during the production of rHSA in presence and absence of molecular chaperones in E. coli have also been studied. CONCLUSION: In the present case, we have filled in the gap in the literature by exploiting the E. coli host system, which is fast-growing and scalable at the low cost of fermentation, as a microbial factory for the enhancement of functional production of rHSA, a crucial protein for therapeutic and biotechnological applications.

Real-Time Tracking the Synthesis and Degradation of Albumin in Complex Biological Systems with a near-Infrared Fluorescent Probe.

In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.

Co-overexpression of Mgat1 and Mgat4 in CHO cells for production of highly sialylated albumin-erythropoietin.

Terminal sialic acids on N-glycan of recombinant human erythropoietin are very important for in vivo half-life, as this glycoprotein has three N-glycosylation sites. N-acetylglucosaminyltransferases I, II, IV, and V (i.e. Mgat1, Mgat2, Mgat4, and Mgat5) catalyze the formation of a glycan antennary structure. These enzymes display different reaction kinetics for a common substrate and generally show low expression in Chinese hamster ovary (CHO) cells. Therefore, genetic control of Mgat expression is an effective method to increase sialic acid contents by enhancing glycan antennarity. To produce highly sialylated albumin-erythropoietin (Alb-EPO), we co-overexpressed the Mgat1 and Mgat4 genes in CHO cells and determined the optimal ratio of Mgat1:Mgat4 gene expression. All transfected cell lines showed increased gene expression of Mgat4, including Mgat1 overexpressing cell line. Sialic acid content of Alb-EPO was highest in co-transfected cells with excess Mgat4 gene, and these cells showed a higher tri- and tetra-antennary structure than control cells. Based on these results, we suggest that co-transfection of the Mgat1 and Mgat4 genes at a ratio of 2:8 is optimal for extension of antennary structures. Also, regulation of Mgat gene expression in the glycan biosynthesis pathway can be a novel approach to increase the terminal sialic acids of N-glycans.