重组人血清白蛋白表达研究进展

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。     

重组人血清白蛋白表达研究进展

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。     

重组人血清白蛋白抗体检测方法的建立及确证

目的：建立一种高灵敏度、高特异性、操作简单快捷、通量高的重组人血清白蛋白（rHSA）抗体检测方法。方法：采用桥连ELISA法,即将rHSA包被于96孔板,加入待测血样及阳性对照,用辣根过氧化物酶标记的rHSA检测,显色读取D_450nm/D_570nm值：用此方法确定临界值、方法灵敏度、精密度、血药浓度对检测方法的影响,再以免疫清除法进行确证。结果：通过桥连ELISA法确定临界值为0．0492,方法灵敏度为352ng／mL,方法板间、板内精密度均小于20％,且血药中的rHSA浓度为20μg／mL时不影响抗体的检测;经免疫清除法可将假阳性样本排除,从而提高了方法的特异性．结论：建立的方法可以准确、快速地检测出rHSA的特异性抗体。     

人血清白蛋白及其重组表达研究进展

人血清白蛋白在医疗方面有广泛的应用,其可作为血容量扩张剂和辅助治疗剂、培养基组分和保护剂、药物制剂、诊断试剂和医疗器械包被剂等。基因重组技术提供了一种规模制备人血清白蛋白的有效途径,并已建立了多种人血清白蛋白的重组表达系统。本文综述了重组人血清白蛋白在细菌、真菌、转基因植物和转基因动物等表达系统中的发展和应用概况。     

人血清白蛋白在乳酸克鲁维酵母中的表达

以乳酸克鲁维酵母(Kluyveromyces lactis,K.lactis)GG799为宿主对人血清白蛋白(HSA)进行分泌表达。以pPIC9k-HSA为模板,采用带有XhoⅠ和NotⅠ酶切位点的引物PCR扩增获得HSA基因,经XhoⅠ和NotⅠ双酶切后插入pKLAC1,构建表达载体pKLAC1-HSA。经SalⅡ线性化后,电击转化K.lactis GG799,用含5 mmol/L乙酰胺的YCB平板筛选阳性转化子。提取基因组DNA,采用PCR方法对转化子鉴定后进行摇瓶发酵。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot分析发酵上清液中的表达产物,并初步分析酵母基础N源(YNB)对HSA在K.lactis GG799中表达的影响。结果表明,HSA成功在K.lactis GG799中分泌表达,表达量为81μg/mL,遗传稳定性好。     

重组人血清白蛋白生产工艺研究进展

人血清白蛋白是一种重要的临床药物 ,人源血浆白蛋白可能会传染病原体将逐步被重组白蛋白所代替。对用巴斯德毕赤酵母生产重组人血清白蛋白的表达系统、发酵方法、分离纯化技术以及重组白蛋白的理化及生理特性进行了综述。     

重组人血清白蛋白在Pichia pastoris中分泌表达影响因素的研究

为获得高产稳产重组人血清白蛋白的Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ细胞株用于高密度发酵,构建了多种分泌表达载体,在Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ中作表达,研究表达载体构建等因素对表达量的影响。发现采用酿酒酵母α性成熟因子前导肽比人血清白蛋白天然前导肽作为分泌信号肽的表达量要高１０％左右。而载体整合方式、整合拷贝数、５’非翻译区的改造和甲醇利用表型等对表达量无规律性影响。筛选到的高表达株经高密度发酵,细     

人重组白蛋白基因在巴斯德毕赤酵母中的高效表达

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.     

重组巴氏毕赤酵母高密度发酵表达rHSA

对基因工程菌Pichiapastoris的摇瓶发酵条件进行了试验 ,并根据摇瓶发酵的优化结果进行了补料分批高密度发酵。在摇瓶发酵时 ,甲醇诱导基因工程菌P .pastoris表达重组人血清白蛋白的发酵周期为 96h ;甲醇的最佳诱导浓度为 1 0g L ;发酵pH范围为 5 72～ 6 5 9;在摇瓶培养时 ,随着接种量的增加 ,虽然目的蛋白表达量缓慢增加 ,但单位细胞光密度的蛋白产率却明显下降 ,符合y =1 2 941x- 0 50 59方程 (线性相关系数r=0 9789) ,其限制性因子很可能为溶氧。在分批发酵 ,接种量为 1 0 %且种子细胞光密度 (OD60 0 )为 2 0左右时 ,细胞生长的延迟期为 2 1 1h左右 ,细胞生长光密度与培养时间的关系模型为 :y =0 7841e0 .2 3 19t(线性相关系数r=0 .993 6 ) ;在补料发酵时细胞干重浓度可达到 1 1 5g L— 1 6 0g L ,在 1 2 0h重组人血清白蛋白表达量最大达到 3 6g L。     

重组人血清白蛋白-干扰素α2b融合蛋白在PichiaPink系统中的表达

目的:比较PichiaPink表达系统和巴斯德毕赤酵母GS115在表达外源蛋白方面的差异,对PichiaPink表达系统的潜在优点进行评价。方法:以重组人血清白蛋白-干扰素α2b融合蛋白(HSA-IFN-α2b)为报告蛋白,构建相关载体,分别转化PichiaPink系统菌株和巴斯德毕赤酵母GS115菌株,诱导HSA-IFN-α2b表达并测定表达水平;通过SDS-PAGE检测HSA-IFN-α2b在PichiaPink系统中的降解程度。结果:PichiaPink系统几乎所有的转化子都可以表达HSA-IFN-α2b,而GS115菌株只有60%的转化子能表达HSA-IFN-α2b;同一种Pink菌株中,整合有高拷贝数表达载体pPink-HC的菌株表达量高于整合有低拷贝数表达载体pPink-LC的菌株;Pink蛋白酶缺陷菌株在复杂培养基(YPD,BMMY)中HSA-IFN-α2b基本没有降解,而在合成培养基(BSM)中降解现象明显。结论:PichiaPink表达系统产生的转化子较GS115菌株产生的转化子易于筛选;PichiaPink系统蛋白酶缺陷菌株可明显减少外源蛋白降解。这些结果为利用PichiaPink表达系统高水平和大规模制备外源蛋白提供了实验依据。     

Comparison of interactions between human serum albumin and silver nanoparticles of different sizes using spectroscopic methods

Three different sizes (15.9 ± 2.1 nm, 26.4 ± 3.2 nm and 39.8 ± 4.0 nm, respectively) of citrate‐coated silver nanoparticles (SNPs) have been synthesized and characterized. The interactions of the synthesized SNPs with human serum albumin (HSA) at physiological pH have been systematically studied by UV‐vis absorption spectroscopy, fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The results indicate that the SNPs can bind to HSA with high affinity and quench the intrinsic fluorescence of HSA. The binding constants and quenching rate constants were calculated. The apparent association constants (K_app) values are 2.14 × 10⁴ M^–1 for 15.9 nm SNP, 1.65 × 10⁴ M^–1 for 26.4 nm SNP and 1.37 × 10⁴ M^–1 for 39.8 nm SNP, respectively. The values of binding constant obtained from the fluorescence quenching data match well with that determined from the absorption spectral changes. These results suggest that the smaller SNPs have stronger interactions to HSA than the larger ones at the same concentrations. Synchronous fluorescence, three‐dimensional fluorescence and CD spectroscopy studies show that the synthesized SNPs can induce slight conformational changes in HSA. Copyright © 2014 John Wiley & Sons, Ltd.     

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (T_m1) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (T_m of ～62°C). When fatty acids are bound to HSA, the structure is stabilized (T_m of ～64–72°C), and prolonged heating (pasteurization at 60°C) results in a heat‐stabilized structural form containing fatty acids (T_m of ～75–80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:62–69, 2015     

Expression of human serum albumin in methylotrophic yeast Pichia pastoris and its structural and functional analysis

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.     

Investigation into the interaction of methylparaben and erythromycin with human serum albumin using multispectroscopic methods

In this paper, the interaction of methylparaben and erythromycin with human serum albumin (HSA) was studied for the first time using spectroscopic methods including Fourier transform infrared (FTIR) spectroscopy and UV absorption spectroscopy in combination with fluorescence quenching under physiological conditions. The binding parameters were evaluated using a fluorescence quenching method. Based on Förster's theory of non‐radiation energy transfer, the binding average distance, r between the donor (HSA) and the acceptor (methylparaben and erythromycin) was evaluated. UV/vis absorption, FTIR, synchronous and 3D spectral results showed that the conformation of HSA was changed in the presence of methylparaben and erythromycin. The thermodynamic parameters were calculated according to the van't Hoff equation and are discussed. The effect of some biological metal ions and site probes on the binding of methylparaben and erythromycin to HSA were further examined. Copyright © 2015 John Wiley & Sons, Ltd.     

Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens

The production of pharmaceutical proteins in plants provides a valuable alternative to other traditional eukaryotic expression systems from economic and safety perspectives. The moss Physcomitrella patens allows the expression and secretion of complex target proteins into a simple aqueous maintenance medium, which facilitates downstream processing by rendering it less complex. To address the question of whether the addition of protein-stabilizing substances enhances the recovery of a target protein secreted into the culture medium, several additives at different concentrations were tested in a small-scale screening system. Although polyvinylpyrrolidone (PVP) and human serum albumin (HSA) showed a significant impact on protein levels, supplementation of the medium with these substances was accompanied by certain limitations in upstream processes, such as foam formation (HSA), and in downstream processes, such as reduced binding efficiency on chromatography columns (PVP), respectively. In order to reap the benefit of the enhancing effect and to avoid the given negative aspects, we developed a new strategy based on the recombinant expression of HSA in plants that are already capable of expressing a target protein. First, we analysed the expression and secretion of recombinant HSA in transiently and stably transformed wild-type (WT) plants. HSA was then co-expressed in Physcomitrella plants transgenic for human vascular endothelial growth factor (VEGF). Even with high expression levels of recombinant human VEGF (rhVEGF), the co-expression of recombinant HSA (rHSA) resulted in 48%-102% higher recovery of the target protein without concomitant negative effects on the upstream process. This strategy enables the enhanced recovery of target protein and does not require the addition of foreign components directly to the culture medium.     

Maximizing recombinant human serum albumin production in a Muts 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^oC 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 mg_rHSA/(g_wcw h)]. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1488–1496, 2014     

引起重组白蛋白电泳检测假性结果的研究

目的:建立SDS-PAGE结合pH值变化对重组人血白蛋白电泳检测假性结果的分析方法。方法:采用还原SDS-PAGE(12.5%)和Native-PAGE(8%～25%),将包含重组人血白蛋白的发酵上清液的pH值分别调为4.0、5.0、6.0、6.5、7.5、8.0、9.0和10.0进行电泳分析。结果:在不同的pH值条件下,重组人血白蛋白会出现不同程度的降解。结论:包含重组人血白蛋白的发酵液中存在不同种类的蛋白酶,导致重组人血白蛋白的假性降解。     

Study of the interaction between ofloxacin and human serum albumin by spectroscopic methods

The binding of ofloxacin (OFLX) to human serum albumin (HSA) was investigated by fluorescence and circular dichroism (CD) techniques. The binding parameters have been evaluated by a fluorescence quenching method. Competitive binding measurements were performed in the presence of warfarin and ibuprofen and suggest binding to the warfarin site I of HSA. The distance r between donor (HSA) and acceptor (OFLX) was estimated according to the Forster's theory of non‐radiatiative energy transfer. CD spectra revealed that the binding of OFLX to HSA induced conformational changes in HSA. Molecular docking was performed and shows that for the lowest energy complex OFLX is located in site I of HSA, which correlate to the competitive binding experiments. Copyright © 2011 John Wiley & Sons, Ltd.     

Interaction of pirprofen enantiomers with human serum albumin.

The interaction of pirprofen enantiomers with human serum albumin (HSA) was investigated by means of high-performance liquid chromatography (HPLC), circular dichroism (CD), and 1H NMR spectroscopy. HPLC experiments indicated that both pirprofen enantiomers were bound to one class of high-affinity binding sites (n(+) = 1.91 +/- 0.13, K(+) = (4.09 +/- 0.64) x 10(5) M-1, n(-) = 2.07 +/- 0.13, K(-) = (6.56 +/- 1.35) x 10(5) M-1) together with nonspecific binding (n'K'(+) = (1.51 +/- 0.21) x 10(4) M-1, n'K'(-) = (0.88 +/- 0.13) x 10(-4) M-1). Slight stereoselectivity in specific binding was demonstrated by the difference in product n(+)K(+) = (0.77 +/- 0.08) x 10(6) M-1 vs. n(-)K(-) = (1.30 +/- 0.21) x 10(6) M-1, i.e., the ratio n(-)K(-)/n(+)K(+) = 1.7. CD measurements showed changes in the binding sites located on the aromatic amino acid side chains (a small positive band at 315 nm and a pronounced negative extrinsic Cotton effect in the region 250-280 nm). The protein remains, however, in its predominantly alpha-helical conformation. The 1H NMR difference spectra confirmed that both pirprofen enantiomers interacted with HSA specifically, most probably with site II on the albumin molecule.