Production Of Human Serum Albumin Research Articles

CRISPR-Cas9 knockout screen informs efficient reduction of the Komagataella phaffii secretome

BackgroundThe yeast Komagataella phaffii is widely used for manufacturing recombinant proteins, but secreted titers of recombinant proteins could be improved by genetic engineering. In this study, we hypothesized that cellular resources could be redirected from production of endogenous proteins to production of recombinant proteins by deleting unneeded endogenous proteins. In non-model microorganisms such as K. phaffii, however, genetic engineering is limited by lack gene annotation and knowledge of gene essentiality.ResultsWe identified a set of endogenous secreted proteins in K. phaffii by mass spectrometry and signal peptide prediction. Our efforts to disrupt these genes were hindered by limited annotation of essential genes. To predict essential genes, therefore, we designed, transformed, and sequenced a pooled library of guide RNAs for CRISPR-Cas9-mediated knockout of all endogenous secreted proteins. We then used predicted gene essentiality to guide iterative disruptions of up to 11 non-essential genes. Engineered strains exhibited a ~20× increase in the production of human serum albumin and a twofold increase in the production of a monoclonal antibody.ConclusionsWe demonstrated that disruption of as few as six genes can increase production of recombinant proteins. Further reduction of the endogenous proteome of K. phaffii may further improve strain performance. The pooled library of secretome-targeted guides for CRISPR-Cas9 and knowledge of gene essentiality reported here will facilitate future efforts to engineer K. phaffii for production of other recombinant proteins and enzymes.

Preserved structure and function of human serum albumin self-folded in the oxidative cytoplasm of Escherichia coli

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.

Purslane (Portulaca oleracea L.) as a novel green-bioreactor for expression of human serum albumin (HSA) gene.

Transgenic plants showed high potential to become a valuable and safe source of bio-compounds that can be used as therapeutics without any require for pooled human blood products. Human serum albumin (HSA) is one of the best-selling pharmaceuticals in the world because it is utilized for treating several acute illnesses, including hypovolemia, burns, and hemorrhage. This work was aimed to investigate the production of recombinant HSA (rHSA) protein in a plant-based expression platform. For this, we used in-planta and tissue culture-based Agrobacterium-mediated transformation (TCBAT) procedures to insert HSA gene into purslane (Portulaca oleracea L.) genome. The purslane seeds and leaves were infected with A. tumefaciens strain LBA4404 containing the HSA gene on pBI121 plasmid, and then regenerated into transgenic plant on MS medium. The qRT-PCR, southern hybridization, western blotting, and ELISA analysis were accomplished to corroborate the insertion and expression of HSA gene in transgenic plantlets. The molecular asses indicated that HSA gene was successfully transferred and expressed in purslane plants using in-planta and TCBAT methods. The first attempt to express rHSA in purslane resulted in a low-level accumulation of the protein in the transgenic plant shoots. Therefore, we used a synthetic 5'UTR (synJ) to enhance HSA transcript stability and translation efficiency. The results suggested that the synJ caused pronounced enhancement of rHSA expression rate. The highest amount of rHSA protein was recorded in transgenic purslane generated by TCBAT method (33.92 ± 4.31µg/g FW).

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

ObjectiveTo improve the yield of recombinant human serum albumin (HSA) in Pichia pastoris by medium optimization and establish the related purification scheme. ResultsA 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. ConclusionMedium 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.

Impacts of Magnetic Immobilization on the Recombinant Proteins Structure Produced in Pichia pastoris System.

Pichia pastoris expression system was introduced with post-translation process similar to higher eukaryotes. Preliminary studies were performed toward process intensification and magnetic immobilization of this system. In this experiment, effects of magnetic immobilization on the structure of recombinant protein were evaluated. P. pastoris cell which express human serum albumin (HSA) was used as a model. The cells were immobilized with various concentrations of APTES coated magnetite nanoparticles. HSA production was done over 5days induction and structure of the product was analyzed by UV-vis, fluorescence, and ATR-FTIR spectroscopy. Second derivative deconvolution method was used to analyze the secondary structure of HSA. P. pastoris cell that were immobilized with 0.5 and 1 mg/mL of nanoparticles were produced HSA with intact structure. But immobilization with 2 mg/mL of nanoparticles resulted in some modifications in the secondary structures (i.e., α-helixes and β-turns) of produced HSA. Based on these data, immobilization of P. pastoris cells with 0.5 or 1 mg/mL of nanoparticles is completely efficient for cell harvesting and has any effect on the structure of recombinant product. These findings revealed that decoration of microbial cells with high concentrations of nanoparticles has some impacts on the structure of secretory proteins.

Engineered Deregulation of Expression in Yeast with Designed Hybrid-Promoter Architectures in Coordination with Discovered Master Regulator Transcription Factor.

Engineered promoters are key components in the cell-factory design, allowing precise and enhanced expression of genes. Promoters having exceptional strength are attractive candidates for designing metabolic engineering strategies for tailoring de novo production strategies that require directed evolution methods by engineering with de novo synthetic biology tools. Here, the custom-designed AOX1 hybrid-promoter architectures in coordination with targeted transcription factors are shown, transcriptionally rewired the expression over methanol-free substrate-utilization pathway(s) and converted methanol-dependent Pichia pastoris alcohol oxidase 1(AOX1) promoter (PAOX1 ) expression into a non-toxic carbon-source-regulated system. AOX1 promoter variants are engineered by replacing specified cis-regulatory DNA elements with synthetic Adr1, Cat8, and Aca2 cis-acting DNA elements for Mxr1, Cat8, and Aca1 binding, respectively. Applications of the engineered-promoters are validated for eGFP expression and extracellular human serum albumin production. The hybrid-promoter architecture designed with single Cat8 cis-acting DNA element deregulates the expression on ethanol. Compared with PAOX1 on methanol, the expression on ethanol is increased with i) PAOX1/Cat8-L3 (designed with single Cat8 cis-acting element) to 74%, ii) PAOX1/Adr1-L3/Cat8-L3 (designed with single- Cat8 and Adr1 cis-acting elements) to 85%, and for further consolidation of deregulated expression iii) PeAOX1 (designed with triplet- Cat8 and Adr1 cis-acting elements) 1.30-fold, at t = 20 h of batch cultivations.

Magnetic Immobilization of Pichia pastoris Cells for the Production of Recombinant Human Serum Albumin.

Magnetic immobilization as a novel technique was used to immobilize recombinant Pichia pastoris (GS115 Albumin) cells to produce human serum albumin (HSA). In this regard, magnetic nanoparticles (MNPs) coated with amino propyl triethoxy silane (APTES) were synthesized. P. pastoris cells were decorated with MNPs via nonspecific interactions. Decorated cells were magneto-responsible and easily harvested by applying an external magnetic field. The efficiency of magnetic immobilization (Ei) for cell removal was in direct relation with the MNP concentration and time of exposure to the magnetic field. By increasing the nanoparticles concentration, cells were harvested in a shorter period. Complete cell removal (Ei ≈ 100) was achieved in ≥0.5 mg/mL of MNPs in just 30 s. HSA is produced in an extremely high cell density (OD ~20) and it is the first time that magnetic immobilization was successfully employed for harvesting such a thick cell suspension. After 5 days of induction the cells, which were immobilized with 0.25 to 1 mg/mL of nanoparticles, showed an increased potency for recombinant HSA production. The largest increase in HSA production (38.1%) was achieved in the cells that were immobilized with 0.5 mg/mL of nanoparticles. These results can be considered as a novel approach for further developments in the P. pastoris-based system.

Statistically Designed Medium Reveals Interactions between Metabolism and Genetic Information Processing for Production of Stable Human Serum Albumin in Pichia pastoris.

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of α–factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

Engineering of alcohol dehydrogenase 2 hybrid-promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol.

The aim of this work is to increase recombinant protein expression in Pichia pastoris over the ethanol utilization pathway under novel-engineered promoter variants (NEPVs) of alcohol dehydrogenase 2 promoter (PADH2 ) through the generation of novel regulatory circuits. The NEPVs were designed by engineering of transcription factor binding sites (TFBSs) determined by in silico analyses and manual curation systematically, by (a) single-handedly replacement of specified TFBSs with synthetic motifs for Mxr1, Cat8, and Aca1 binding, and synthetic TATA-box integration; and, (b) nucleosome optimization. PADH2-Cat8-L2 and PADH2-Cat8-L1 designed by the integration of synthetic Cat8 binding sites were superior, and then PADH2-NucOpt . Compared to that with PADH2 at t = 20 hr of the fermentations, PADH2-Cat8-L2 allowed the highest increase in enhanced green fluorescent protein expression as 4.8-fold on ethanol and 3.8-fold on methanol; and, PADH2-NucOpt upregulated the expression 1.5-fold on ethanol and enhanced 3.2-fold on methanol. Using the superior two tools, Cat8-L2 and NucOpt, we designed PADH2-NucOpt-Cat8-L2 . With PADH2-NucOpt-Cat8-L2 , the expression in the fermentation of ethanol was upregulated 3.7-fold that is distinctly higher than that with PADH2-NucOpt but lower than that with PADH2-Cat8-L2 ; while on methanol compared to that with PADH2 , the expression was enhanced 8.8-fold. Extracellular recombinant human serum albumin production was also studied with PADH2-Cat8-L2 and PADH2-NucOpt , and average recombinant human serum albumin yield (YP/X ) on ethanol was 1.13 and 0.38 mg/gWCW, respectively; whereas with PADH2 , YP/X was 0.26 mg/gWCW . We conclude that as upregulation of transcription and enhanced expression correlate with the sequence of synthetic motifs and their location in the hybrid-promoter architectures of NEPVs in coordination with trans-acting factors, which are the design parameters in the engineering of binding sites; the NEPVs generated promising recombinant protein production platforms with a high impact on industrial scale production processes, as well as would open up new avenues for research in P. pastoris.

Twisted intramolecular charge transfer (TICT) based fluorescent probe for lighting up serum albumin with high sensitivity in physiological conditions.

Accurate detection of human serum albumin (HSA) in biological samples is quite meaningful for early disease diagnosis and treatment. Herein, a novel fluorescent probe 1-ethyl-4-[2-[4-(diethylamino)-2-hydroxyphenyl]ethenyl]]-pyridinium salt (DEHP) was developed for HSA determination. The inherent fluorescence of DEHP is essentially negligible at physiological conditions assigned to the well-developed twisted intramolecular charge transfer (TICT) protocol. An intriguing fluorescence light up is triggered as the addition of HSA, on account of the inhibited TICT procedure when DEHP enters the hydrophobic cavity of protein HSA. This combination leads to a turn on fluorescent response for HSA with a detection limit of 4.8 nM. After an overall investigation, it has been proved that the strong binding between DEHP and HSA is specific-site-related. In additional, the probe implies a great potential to assist clinical diagnosis due to the usage in actual serum detection. Cell imaging also shows that the probe is expected to monitor HSA production process at cell level.

Biosensor-assisted engineering of a high-yield Pichia pastoris cell-free protein synthesis platform.

Cell-free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell-free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor, we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a three-fold increase in recombinant protein yield compared with those from the wild-type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg ml -1 . Therefore, this study not only adds to the growing number of CFPS systems from diverse organisms but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.

New insights into the altered binding capacity of pharmaceutical-grade human serum albumin: site-specific binding studies by induced circular dichroism spectroscopy

The ADMET profile of drugs is strongly affected by human serum albumin (HSA), due to its leading role as carrier of poorly soluble compounds in plasma; a critical assessment of the binding capacity of HSA and the evaluation of binding competition between drugs are therefore pivotal for a reliable pharmacokinetic and pharmacodynamic characterization. In clinical practice, a potential source of impairment in the binding properties of HSA is the use of octanoate and N-acetyltryptophan as stabilizers during the production of pharmaceutical-grade HSA for infusion (i-HSA), which is currently administered in the treatment of a growing range of pathological conditions. The peculiar sensitivity of circular dichroism (CD) spectroscopy towards the stereochemical features of high-affinity binding events is herein exploited to achieve a site-specific assessment of the effect of stabilizers on the binding properties of i-HSA. The binding affinity and capacity of fatty-acid-free HSA towards site-selective induced circular dichroism (ICD) markers for the three high-affinity binding sites of HSA was compared to that of i-HSA submitted to ultrafiltration and dialysis to remove both stabilizers. Results showed a considerable impairment of the binding capacity of i-HSA at site II and a relatively lower influence on the binding properties of site I. Ultrafiltration proved to be ineffective in depleting octanoate, while the proposed dialysis protocol, which involves a pH-induced reversible unfolding of the protein, resulted in a total clearance of both stabilizers, confirmed by the full restoration of the binding properties of HSA at all binding sites. The outcomes of this study proved that CD spectroscopy is a suitable technique to evaluate the binding properties of i-HSA, ensuring an assessment of the availability of the binding sites and the possibility of monitoring the clearance of stabilizers. Eventually, the proposed method for their depletion might constitute a connection bridge between albumin in vitro studies and its clinical applications.

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

BackgroundHuman 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.ResultsIn 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.ConclusionIn 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.

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

BackgroundPichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell’s environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions.ResultsIn this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions.Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein.ConclusionIn this work, we formulated a dynamic genome scale metabolic model of Pichia pastoris that yields realistic metabolic flux distributions throughout dynamic cultivations. The model can be calibrated with experimental data to rationally propose genetic and process engineering strategies to improve the performance of a P. pastoris strain of interest.

Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma.

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.

SIMULTANEOUS LOADING OF 5-FLOROURACIL AND SPIONS IN HSA NANOPARTICLES: OPTIMIZATION OF PREPARATION, CHARACTERIZATION AND IN VITRO DRUG RELEASE STUDY

Objective(s): Over the past two decades, considerable interest has been focused on utilizing biocompatible magnetic nanoparticles (MNPs) for biomedical applications. In this study, production of human serum albumin (HSA) nanoparticles using desolvation technique that were simultaneous loaded with high amounts of superparamagnetic iron oxide nanoparticles (SPIONs) and 5-flourouracil (5-FU) was investigated. Materials and Methods: 5-FU loading (%) and SPIONs entrapment efficiency (%) were optimized using response surface methodology (RSM). The design expert software used to analyse the interactive effects of pH, 5-FU and SPIONs concentrations. Results:The optimum conditions found to be pH of 8.2, drug concentration of 1.5 mg/ml and SPIONs concentration of 2.79 mg/ml. Under the mentioned optimum conditions, particles with the size of 111.8 nm, zeta potential of -37.1 mV, 5-FU loading of 15.8% and SPIONs entrapment efficiency of 41.1% were obtained. In vitro cumulative release of 5-FU from the nanoparticles was evaluated in phosphate buffer saline (pH 7.4, 37 °C). Results indicated that 85% of the 5-FU released during 95 h, which revealed a sustained release profile. In addition, Vibrating Sample Magnetometer (VSM) analyses confirmed the superparamagnetic properties of magnetic albumin nanoparticles manufactured under the optimum conditions. Conclusion: According to the findings,SPIONs and 5-FU loaded HAS nanoparticles arepromising for use as novel targeted delivery system due to proper magnetic and drug release behaviours.

Human albumin solution for patients with cirrhosis and acute on chronic liver failure: Beyond simple volume expansion.

To provide an overview of the properties of human serum albumin (HSA), and to review the evidence for the use of human albumin solution (HAS) in critical illness, sepsis and cirrhosis. A MEDLINE search was performed using the terms "human albumin", "critical illness", "sepsis" and "cirrhosis". The references of retrieved articles were reviewed manually. Studies published between 1980 and 2014 were selected based on quality criteria. Data extraction was performed by all authors. HSA is the main plasma protein contributing greatly to its oncotic pressure. HSA demonstrates important binding properties for endogenous and exogenous toxins, drugs and drug metabolites that account for its anti-oxidant and anti-inflammatory properties. In disease states, hypoalbuminaemia is secondary to decreased HSA production, increased loss or transcapillary leakage into the interstitial space. HSA function can be also altered in disease with reduced albumin binding capacity and increased production of modified isoforms. HAS has been used as volume expander in critical illness, but received criticism due to cost and concerns regarding safety. More recent studies confirmed the safety of HAS, but failed to show any survival benefit compared to the cheaper crystalloid fluids, therefore limiting its use. On the contrary, in cirrhosis there is robust data to support the efficacy of HAS for the prevention of circulatory dysfunction post-large volume paracentesis and in the context of spontaneous bacterial peritonitis, and for the treatment of hepato-renal syndrome and hypervolaemic hyponatraemia. It is likely that not only the oncotic properties of HAS are beneficial in cirrhosis, but also its functional properties, as HAS replaces the dysfunctional HSA. The role of HAS as the resuscitation fluid of choice in critically ill patients with cirrhosis, beyond the established indications for HAS use, should be addressed in future studies.

Production of transgenic cattle highly expressing human serum albumin in milk by phiC31 integrase-mediated gene delivery

Transgenic cattle expressing high levels of recombinant human serum albumin (HSA) in their milk may as an alternative source for commercial production. Our objective was to produce transgenic cattle highly expressing HSA in milk by using phiC31 integrase system and somatic cell nuclear transfer (SCNT). The mammary-specific expression plasmid pIACH(-), containing the attB recognition site for phiC31 integrase, were co-transfected with integrase expression plasmid pCMVInt into bovine fetal fibroblast cells (BFFs). PhiC31 integrase-mediated integrations in genome of BFFs were screened by nested inverse PCR. After analysis of sequence of the PCR products, 46.0% (23/50) of the both attB-genome junction sites (attL and attR) were confirmed, and four pseudo attP sites were identified. The integration rates in BF3, BF11, BF19 and BF4 sites were 4.0% (2/50), 6.0% (3/50), 16.0% (8/50) and 20.0% (10/50), respectively. BF3 is located in the bovine chromosome 3 collagen alpha-3 (VI) chain isomer 2 gene, while the other three sites are located in the non-coding region. The transgenic cell lines from BF11, BF19 and BF4 sites were used as donors for SCNT. Two calves from transgenic cells BF19 were born, one died within a few hours after birth, and another calf survived healthy. PCR and Southern blot analysis revealed integration of the transgene in the genome of cloned calves. The nested reverse PCR confirmed that the integration site in cloned calves was identical to the donor cells. The western blotting assessment indicated that recombinant HSA was expressed in the milk of transgenic cattle and the expression level was about 4-8 mg/mL. The present study demonstrated that phiC31 integrase system was an efficient and safety gene delivery tool for producing HSA transgenic cattle. The production of recombinant HSA in the milk of cattle may provide a large-scale and cost-effective resource.

Maximizing recombinant human serum albumin production in a Mut(s) Pichia pastoris strain.

Human serum albumin (HSA) is a cysteine rich molecule that is most abundant in human blood plasma. To remain viable in the market due to lower marketing costs for HSA, it is important to produce a large quantity in an economical manner by recombinant technology. The objective of this study was to maximize recombinant HSA (rHSA) production using a Mut(s) Pichia pastoris strain by fermentation process optimization. We evaluated the impact of process parameters on the production of rHSA, including induction cell density (wet cell weight, g/L) and the control of specific growth rate at induction. In this study, we demonstrated that induction cell density is a critical factor for high level production of rHSA under controlled specific growth rate. We observed higher specific productivities at higher induction cell densities (285 g/L) and at lower specific growth rates (0.0022-0.0024/h) during methanol induction phase, and achieved the broth titer of rHSA up to 10 g/L. The temperature shift from 24 to 28(o) C was effective to control the specific growth rate at low level (≤0.0024/h) during methanol induction phase while maintaining high specific productivity [0.0908 mgrHSA /(gwcw h)].

Expression and purification of recombinant human serum albumin from selectively terminable transgenic rice

Human serum albumin (HSA) is widely utilized for medical purposes and biochemical research. Transgenic rice has proved to be an attractive bioreactor for mass production of recombinant HSA (rHSA). However, transgene spread is a major environmental and food safety concern for transgenic rice expressing proteins of medical value. This study aimed to develop a selectively terminable transgenic rice line expressing HSA in rice seeds, and a simple process for recovery and purification of rHSA for economical manufacture. An HSA expression cassette was inserted into a T-DNA vector encoding an RNA interference (RNAi) cassette suppressing the CYP81A6 gene. This gene detoxifies the herbicide bentazon and is linked to the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) cassette which confers glyphosate tolerance. ANX Sepharose Fast Flow (ANX FF) anion exchange chromatography coupled with Butyl Sepharose High Performance (Butyl HP) hydrophobic interaction chromatography was used to purify rHSA. A transgenic rice line, HSA-84, was obtained with stable expression of rHSA of up to 0.72% of the total dry weight of the dehusked rice seeds. This line also demonstrated high sensitivity to bentazon, and thus could be killed selectively by a spray of bentazon. A two-step chromatography purification scheme was established to purify the rHSA from rice seeds to a purity of 99% with a recovery of 62.4%. Results from mass spectrometry and N-terminus sequencing suggested that the purified rHSA was identical to natural plasma-derived HSA. This study provides an alternative strategy for large-scale production of HSA with a built-in transgene safety control mechanism.