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1.
人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。  相似文献   

2.
重组人血清白蛋白在Pichia pastoris中的表达与纯化   总被引:8,自引:0,他引:8  
为实现重组人血清白蛋白(rHSA)的开发,对构建的酵母工程菌Pichia pastoris GS115/HSA进行了表达条件的优化,摇瓶中将表达rHSA的量提高到150mg/L。经中空纤维柱浓缩、Phenyl-Sepharose分离和抗HSA-Sepharose亲和层析纯化获得电泳纯的重组人血清白蛋白。  相似文献   

3.
重组人血清白蛋白表达研究进展   总被引:1,自引:0,他引:1  
本文综述了重组人血清白蛋白在细菌,酵母,植物和动物等表达系统中表达的研究进展。用酵母表达系统,尤其是毕赤酵母,表达的重组白蛋白产量高且提取工艺简单,是其产业化最具有前途的表达系统。同时在转基因动物、植物中培养表达也具有诱人的前景。  相似文献   

4.
An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.  相似文献   

5.
人重组白蛋白基因在巴斯德毕赤酵母中的高效表达   总被引:10,自引:0,他引:10  
The yeast Pichia pastoris was transformed by the multi\|copy Pichia expression vector that can express secreted human albumin.The high level expression of cell line was selected after screening.The expression of human recombinant albumin in Pichia pastoris induced by different methods were compared.The retio of secreted human albumin is 80% in total secreted proteins and the expression level reaches as high as is 10g/L.  相似文献   

6.
The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.  相似文献   

7.
Recombinant protein expression in Pichia pastoris   总被引:96,自引:0,他引:96  
The methylotrophic yeast Pichia pastoris is now one of the standard tools used in molecular biology for the generation of recombinant protein. P. pastoris has demonstrated its most powerful success as a large-scale (fermentation) recombinant protein production tool. What began more than 20 years ago as a program to convert abundant methanol to a protein source for animal feed has been developed into what is today two important biological tools: a model eukaryote used in cell biology research and a recombinant protein production system. To date well over 200 heterologous proteins have been expressed in P. pastoris. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe's value and power in commercial and research labs alike.  相似文献   

8.
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 Muts 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 28oC 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)]. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1488–1496, 2014  相似文献   

9.
Crystal structure of human serum albumin at 2.5 A resolution.   总被引:8,自引:0,他引:8  
A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.  相似文献   

10.
The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs) leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA) produced in Oryza sativa (Asian rice) (OsrHSA) from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA) and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae). The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC), reversed-phase high-performance liquid chromatography (RP-HPLC) and capillary electrophoresis (CE). Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS). The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD) and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which correlated well with the degree of arginine/lysine glycation. The extensive glycation of OsrHSA from multiple suppliers may have further implications for the use of OsrHSA as a therapeutic product.  相似文献   

11.

Background

As the most abundant protein in the blood, human serum albumin (HSA) plays an important role in maintaining plasma oncotic pressure and fluid balance between the body's compartments. HSA is thus widely used in the clinic to treat diseases. However, the shortage of and safety issues arising from using plasma HSA (pHSA) underscore the importance of recombinant HSA (rHSA) as a promising substitute for pHSA.

Scope of review

Here, we review the production of rHSA, from expression to downstream processing, and highlight the scalability and cost-effectiveness of the two main expression platforms. We also discuss the biosafety of commercially available pharmaceutical rHSA with respect to impurities and contaminants, followed by an analysis of recent progress in preclinical and clinical trials. We emphasise the challenges of producing pharmaceutical-grade rHSA.

Major conclusions

rHSA can be highly expressed in various hosts and seems to be identical to pHSA. rHSA generated from yeast appears to be as efficient and safe as pHSA in a series of preclinical and clinical trials, whereas rHSA from rice seeds exhibits great potential for more cost-effective production. Cost-effective products with no adverse effects will likely play a vital role in future human therapeutics.

General significance

Our understanding of pharmaceutical-grade rHSA production has improved with respect to expression hosts, biochemical properties, downstream processing, and the detection and removal of impurities. However, due to the large dosages required for clinical applications, the production of sufficient quantities of rHSA still presents challenges. This article is part of a Special Issue entitled Serum Albumin.  相似文献   

12.
13.
Banerjee H  Verma M 《Plasmid》2000,43(2):181-183
Certain yeast strains secrete a protein toxin, which inhibits the growth of sensitive pathogens and yeasts. Studies have shown that production of the toxin is dependent on presence of linear, double-stranded DNA plasmids in the killer yeasts. In the yeast Pichia pastoris, two linear double-stranded DNA plasmids have been identified. In the present study, the search for toxin-producing capability in P. pastoris has been conducted. No killer activity could be detected when 14 different indicator strains were tested.  相似文献   

14.
Glycosylation engineering in yeast: the advent of fully humanized yeast   总被引:2,自引:0,他引:2  
Yeasts have been extensively used as model organisms to elucidate cellular processes and their mechanism in lower eukaryotes. Consequently, a large number of powerful genetic tools have been developed to engineer yeast and improve its utility. These tools and the development of efficient large-scale fermentation processes have made recombinant protein expression in yeast an attractive choice. However, for the production of glycoproteins for human use, native high-mannose yeast glycosylation is not suitable and therefore represents a major limitation for yeast based protein expression systems. Over the last two decades several groups have attempted to overcome this problem, yet with limited success. Recently however, major advances in the glycoengineering of the yeast Pichia pastoris, have culminated in the production of fully humanized sialylated glycoproteins.  相似文献   

15.
The methylotrophic yeast, Pichia pastoris, is an important organism used for the production of therapeutic proteins. However, the presence of fungal-like glycans, such as those containing β-mannose (Man) linkages, can elicit an immune response or bind to Man receptors, thus reducing their efficacy. Recent studies have confirmed that P. pastoris has four genes from the β-mannosyl transferase (BMT) family and that Bmt2p is responsible for the majority of β-Man linkages on glycans. While expressing recombinant human erythropoietin (rhEPO) in a developmental glycoengineered strain devoid of BMT2 gene expression, cross-reactivity was observed with an antibody raised against host cell antigens. Treatment of the rhEPO with protein N-glycosidase F eliminated cross-reactivity, indicating that the antigen was associated with the glycan. Thorough analysis of the glycan profile of rhEPO demonstrated the presence of low amounts of α-1,2-mannosidase resistant high-Man glycoforms. In an attempt to eliminate the α-mannosidase resistant glycoforms, we used a systemic approach to genetically knock-out the remaining members of the BMT family culminating in a quadruple bmt2,4,1,3 knock-out strain. Data presented here conclude that the additive elimination of Bmt2p, Bmt3p and Bmt1p activities are required for total abolition of β-Man-associated glycans and their related antigenicity. Taken together, the elimination of β-Man containing glycoforms represents an important step forward for the Pichia production platform as a suitable system for the production of therapeutic glycoproteins.  相似文献   

16.
The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a beta-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of DeltaGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled.  相似文献   

17.
The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.  相似文献   

18.
The hemoflavoenzyme cellobiose dehydrogenase (CDH) from the white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. After 4 days of cultivation in the induction medium, the expression level reached 1800 U/L (79 mg/L) of CDH activity, which is considerably higher than that obtained previously for wild-type CDH (wtCDH) and recombinant CDH (rCDH) produced by P. chrysosporium. Analysis with SDS-PAGE and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with an approximate molecular mass of 100 kDa, which was identified as rCDH by Western blotting. The absorption spectrum of rCDH shows that the protein contains one flavin and one heme cofactor per protein molecule, as does wtCDH. The kinetic parameters for rCDH using cellobiose, ubiquinone, and cytochrome c, as well as the cellulose-binding properties of rCDH were nearly identical to those of wtCDH. From these results, we conclude that the rCDH produced by Pichia pastoris retains the catalytic and cellulose-binding properties of the wild-type enzyme, and that the Pichia expression system is well suited for high-level production of rCDH.  相似文献   

19.
The stable strain of methylotrophic yeast Pichia pastoris secreting human serum albumin into cultural medium was obtained. Optimal conditions for expression of the protein were determined. We characterized the recombinant protein by mass spectrometry and circular dichroism and analyzed its catalytic activity.  相似文献   

20.
Na,K-ATPase is a crucial enzyme for ion homeostasis in human tissues. Different isozymes are produced by assembly of four alpha- and three beta-subunits. The expression of the alpha3/beta1 isozyme is confined to brain and heart. Its heterologous production has so far never been attempted in a lower eukaryote. In this work we explored whether the methylotrophic yeast Pichia pastoris is capable of expressing the alpha3/beta1 isoform of human Na,K-ATPase. cDNAs encoding the alpha(3) and the beta(1)-subunits were cloned under the control of the inducible promoter of Pichia pastoris alcohol oxidase 1. Pichia pastoris could express the single alpha3- and beta1-subunits and even coexpress them after methanol induction. beta1-subunit was produced as a major 44-kDa glycosylated polypeptide and alpha3 as a 110-kDa unglycosylated polypeptide. Expression at the plasma membrane was limited in shaking flask cultures but by cultivating P. pastoris cells in a fermenter there was a 10-fold increase of the number of ouabain binding sites per cell. The exported enzyme was estimated to be about 0.230 mg L(-1) at the end of a bioreactor run. Na,K-ATPase proved active and the dissociation constant of the recombinant enzyme-ouabain interaction was determined.  相似文献   

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