乳腺个体表达系统的重组蛋白质纯化

近年来,利用转基因动物乳腺生产重组蛋白质发展迅速。在研究小鼠模型基础上,1990年春,5只生产人a一抗胰蛋白酶的转基因羊在英国诞生。乳中蛋白质表达水平已达30g／L。迄今已稳定遗传三代,并进人临床实验’“。同年在荷兰诞生了第一头生产人乳铁蛋白的转基因牛,并繁殖20多头。对于这些产奶量极高的动物来说,从其乳中获取重组蛋白,获益是巨大的。目前利用转基因动物做为生物反应器生产重组蛋白已引起广泛兴趣。然而乳作为生产重组蛋白质的载体,特别是其本身含有许多蛋白质,增加了纯化的难度,这方面的研究目前尚不多。本文对研究现…     

外源蛋白在大肠杆菌中的可溶性表达策略

长期以来,大肠杆菌一直是表达外源蛋白的首选表达系统. 但由于外源蛋白在表达过程中容易被宿主细胞蛋白酶降解或者形成包涵体,其应用受到了限制. 本文综述了在大肠杆菌中表达可溶外源蛋白的策略和进展,以期提高具有生物活性的外源基因的表达水平.     

人可溶性CD105蛋白的真核表达及纯化

目的建立一种简便、高效的人可溶性CD105(soluble CD105,sCD105)蛋白的真核表达及纯化方法。方法人工合成胞外区和信号肽区域的DNA片段,在其信号肽前端加入Kozak sequence(GCACCATGG)序列,促进蛋白表达,同时在其C-末端6×His前加入K(AAG)氨基酸,促进6×His的暴露有助于后期纯化,构建真核表达质粒pcDNA3. 4-sCD105。通过瞬时转染的方法将重组质粒转染293F悬浮细胞,收集细胞上清液,利用His-trap EXCEL柱通过两步纯化法进行纯化。将纯化获得的sCD105蛋白超滤浓缩后,进行10%SDS-PAGE、Western blot及ELISA检测。结果在20 mL(1×10~6个/mL)293F悬浮细胞中表达及纯化后,获得纯度 95%的sCD105蛋白3 mL,浓度为27. 83 mg/L,获得率高达4. 17 mg/L。结论成功建立了一种简便高效的sCD105蛋白表达及纯化的方法,为后期蛋白功能的研究及相应治疗或诊断性抗体的开发奠定了基础。     

融合蛋白GST-PADI4可溶性表达条件的优化及纯化

目的优化融合蛋白GST-PADI4的可溶性表达条件,并对蛋白进行纯化。方法对融合蛋白GST-PADI4工程菌的诱导温度(16、20、25、30、37℃)、诱导剂IPTG浓度(0.05、0.1、0.2、0.5、1 mmol/L)、诱导时菌液A600值(0.2、0.4、0.6、0.8、1.0)、诱导时间(8、12、16、20、24 h)进行优化,分析融合蛋白GST-PADI4的可溶性表达情况;按优化的表达条件进行大量表达后,采用Sepharose 4B亲和层析柱对融合蛋白进行纯化。结果在16℃,菌液A600值约为0.4时,以0.1 mmol/L IPTG诱导12 h,融合蛋白GST-PADI4有较高的可溶性表达;纯化的融合蛋白纯度为91%。结论优化了融合蛋白GST-PADI4的可溶性表达条件,并获得了高纯度的可溶性融合蛋白,为后续研究PADI4蛋白的功能奠定了基础。     

GST-pulldown技术在蛋白质相互作用中的应用

蛋白间相互作用在细胞的生命活动中起着关键作用,GST-pull down技术被广泛用于体外验证蛋白间的直接相互作用。GST-pull down技术既可用来验证已知蛋白间的相互作用,也可用来寻找与已知蛋白相互作用的未知蛋白。虽然该技术在体外验证蛋白间的相互作用上有着较强的特异性,但下结论时仍需慎重。本文就GST-pull down技术的原理及其应用及该技术在应用中需要注意的部分问题进行综述。     

HBV PreS2-MBP融合蛋白质粒构建及在大肠杆菌中的表达

目的在大肠杆菌pMALP2x系统中表达HBVPreS2MBP融合蛋白,并对其进行鉴定和纯化。方法利用DNA重组技术,将全长的HBVPreS2蛋白基因克隆至原核表达载体pMALP2x中,转化大肠杆菌DH5α,IPTG诱导表达。表达产物经SDSPAGE和Westernblot检测,并经阴离子交换层析、AmyloseResin亲和层析纯化。结果成功构建了重组载体pMALP2x/S2,诱导蛋白为MBP标记的可溶性的PreS2MBP融合蛋白,具有良好的抗原性,可经AmyloseResin亲和层析纯化。结论pMALP2x系统可成功表达PreS2MBP融合蛋白。     

GST-AEP融合蛋白的表达、纯化和鉴定

目的利用GST融合基因表达系统表达GST-AEP融合蛋白,并进行纯化及鉴定。方法IPTG诱导重组质粒pGEX-4T-1/AEP在大肠杆菌BL21(DE3)中表达,溶菌酶裂解后,采用GST蛋白纯化系统进行纯化,所得产物进行12%SDS-PAGE及Western blot鉴定。结果大肠杆菌经诱导,高效表达出相对分子质量约34000的蛋白,与GST-AEP融合蛋白相符。Western blot结果显示该蛋白能够被兔抗GST多克隆抗体识别。结论已成功表达并纯化了融合蛋白,为更深入研究AEP的结构和功能奠定了基础。     

PTD-SARA融合蛋白的表达、纯化及鉴定

目的在大肠杆菌中表达PTD-SARA融合蛋白,并对其进行纯化和鉴定。方法使用大肠杆菌偏爱密码子合成PTD-SARA全长基因,将其克隆入载体pQE-30中,构建重组表达质粒pQE-30-PTD-SARA,转化感受态大肠杆菌M15,IPTG诱导表达。表达产物经Ni2+-NTA亲和层析纯化后,进行Western blot鉴定及N-端测序。结果重组表达质粒pQE-30-PTD-SARA经双酶切及测序鉴定证明构建正确。1.0mmol/LIPTG诱导4h,目的蛋白的表达量最高,约占菌体总蛋白的25%,主要以包涵体形式存在。纯化的融合蛋白纯度为94%,浓度为0.2mg/ml,且具有良好的反应原性,N-端有14个氨基酸。结论已成功表达并纯化了PTD-SARA融合蛋白,为其功能的研究奠定了基础,也为临床防治肾脏纤维化提供了一个新的策略。     

重组基因工程菌几丁质酶C的可溶性表达研究

在不同培养温度条件下对产几丁质酶C的基因工程菌BL21（DE3）进行诱导,使其表达可溶性蛋白.随后在较佳培养温度诱导条件的基础上向培养基中添加不同浓度的甘油,甘氨酸,山梨醇/甜菜碱等成分来更进一步的提高几丁质酶C的可溶性表达.其结果是在25℃诱导条件下,以添加2g/L葡萄糖的LB培养基作为基础培养基进行培养,在基础培养基中添加3g/L甘油的总酶活最高,达到了18.17 U/mL,较之仅含2g/L葡萄糖的LB培养基在37℃及25℃诱导培养条件下酶活力分别提高了41.6％和20.3％;添加0.3％甘氨酸约90％的可溶性几丁质酶表达到了胞外,胞外酶活达到14.68 U/mL;添加0.5 M山梨醇/2.5 mM甜菜碱工程菌胞内酶活达到最高,为8.43 U/mL.结果表明适当降低工程菌诱导表达时的培养温度提高了几丁质酶C的可溶性表达,在较佳温诱导表达的基础上向培养基中添加不同浓度的甘油,甘氨酸,山梨醇/甜菜碱更进一步的提高了几丁质酶C的可溶性表达.     

人溶菌酶-木聚糖酶融合基因在毕赤酵母中的表达

目的在毕赤酵母中表达人溶菌酶(Human lysozyme,hLY)-木聚糖酶(Xylanases,XynⅡ)融合基因。方法通过PCR技术将hLY基因与XynⅡ基因连接,中间插入肠激酶识别位点序列;将其克隆至载体pPIC9K上,构建重组表达质粒pPIC9K-XynⅡ-EKsite-hLY,经SacⅠ线性化后,电转化毕赤酵母GS115,通过G418抗性筛选得到高拷贝转化子,PCR鉴定为阳性的克隆用甲醇进行诱导;表达的融合蛋白经Sephadex G-75凝胶柱层析纯化后,用肠激酶酶切,分别采用改良舒加法和DNS法测定hLY和XynⅡ的活性。结果获得的融合基因序列与理论序列完全一致;重组表达质粒构建正确;融合蛋白的hLY和XynⅡ活性分别为170和158 U/ml,经肠激酶酶切后,hLY的活性为520 U/ml,XynⅡ的活性达244 U/ml。结论已在毕赤酵母中成功表达了XynⅡ-EKsite-hLY融合基因,经肠激酶酶切后的目的蛋白活性均有较大提高。     

Expression of intein‐tagged fusion protein and its applications in downstream processing

The conventional methods of downstream purification of a recombinant protein are not only complicated and delicate but time consuming, and need to be improved. Since the intein, the protein splicing element, was discovered, this self‐cleaving element has been exploited and applied to the purification of recombinant proteins which could significantly simplify the purification procedure. Intein has the unique property that when it is combined with an affinity tag, it enables a target protein to be purified in a single chromatographic step. This review elucidates the properties of intein (the mechanism that unravels the intein‐based protein splicing), the advantages of an intein affinity expression system, the progress of intein‐based protein purification procedures, and recent advances in the applications of intein. Further development of the intein‐based purification system may lead to the applications of this system to industrial‐scale production of recombinant proteins. Copyright © 2009 Society of Chemical Industry     

Plasmid stability in a recombinant S cerevisiae strain secreting a bifunctional fusion protein

The recombinant Saccharomyces cerevisiae strain, YPB‐G, producing and secreting Bacillus subtilis α‐amylase and Aspergillus awamori glucoamylase as a fusion protein yielded efficient utilisation of starch. A segregated population balance model has been used to determine the probability of plasmid loss and plasmid copy number. The kinetics of cell growth and product (fusion protein) formation were based on a genetically structured model. The predictions were compared with the experimental observations obtained for the unstable recombinant S cerevisiae cells in a 1.5 dm^?3 batch bioreactor with 30 g dm³ initial starch under non‐aerated conditions. The main advantage of the present model is that three different genetic classes were defined on the basis of the existence of plasmid and of the expression of the enzymes, ie cells containing plasmids and expressing the gene product, x₁; cells containing plasmids and but not expressing the gene product, x₂; and cells without plasmids, x₃. It is confirmed by this model that the cells without plasmids outgrow and dominate in the fermentation medium (2.27 g dm^?3 vs 0.51 g dm^?3) as more and more glucose becomes available by the degradation of starch. © 2001 Society of Chemical Industry     

猪圆环病毒2b亚型Cap融合蛋白的原核表达及其免疫原性分析

目的原核表达猪圆环病毒2b亚型(porcine circovirus subtype 2b,PCV2b)Cap融合蛋白,并分析其免疫原性。方法以PCV2毒株(CAU0673)基因组为参考序列,His-Sumo-PCV2b-Cap-pUC57重组质粒为模板,设计特异性引物,PCR扩增目的片段;将其产物克隆至pMAL-C4x载体中,构建pC4x-PCV2b-Cap重组质粒,转化至大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物进行直链淀粉树脂纯化。SDS-PAGE分析目的蛋白的可溶性,Western blot检测其反应原性;以纯化的融合蛋白免疫BALB/c小鼠,Western blot检测小鼠血清抗体特异性。结果经双酶切及测序鉴定,重组质粒pC4x-PCV2b-Cap构建正确;融合蛋白MBP-Cap(matlose binding protein Cap)最适诱导条件为1 mmol/L IPTG 37℃诱导6 h,其以可溶性形式存在,相对分子质量约为81000,与PCV2b阳性猪血清、兔抗MBPtag多克隆抗体及免疫小鼠抗血清均可发生特异性反应。结论成功构建了pC4x-PCV2b-Cap重组表达载体,获得了可溶性的MBP-Cap蛋白,其纯化蛋白具有较好的免疫原性。本文为PCV2b诊断试剂盒的研发奠定了理论基础。     

Production and purification of a fused recombinant protein gp-36 (HIV-2) from Escherichia coli

A fragment of the gp-36 gene of the Human Immunodeficiency Virus type 2 (HIV-2) was fused to a stabilizer sequence, which encodes for the first N-terminal 58 amino acids of the human interleukin-2. The fused protein was expressed under the control of the tryptophan promoter in Escherichia coli, and expressed as 20% of the total cellular protein. Transmission electron microscopy indicated that the fusion protein formed cytoplasmic insoluble inclusion bodies. Inclusion bodies were semipurified by a wash pellet cell procedure, rendering a material with a purity higher than 70% by SDS–polyacrylamide gel electrophoresis. After solubilization with urea, this preparation was further purified by gel-filtration chromatography up to 95% purity.     

人M-CSF与SCF融合蛋白的构建及其在昆虫细胞中的表达

应用重组DNA技术构建M CSF与SCF的融合基因并将其克隆于昆虫杆状病毒转移载体 pVL13 92中 ,通过与野生型苜蓿夜蛾核型多角体病毒 (AcNPV)DNA共转染草地夜蛾细胞Sf9,融合基因插入AcNPV基因组。重组病毒感染单层Sf9细胞后 ,表达产物分泌到胞外培养液中 ,用MTT比色法和TF 1细胞株可检测到表达产物与IL 3的协同效应。上述研究为开发具有应用价值的新型细胞融合因子奠定了基础     

Improved method for the isolation and purification of water‐soluble polyphosphazenes

We report in this article an improved procedure to isolate and purify representative water‐soluble polyphosphazenes that dramatically reduces the time and equipment involved, while maintaining or exceeding the yields and purity reported in the literature for these polymers obtained using dialysis methods. This technique takes advantage of the phase transition behavior exhibited by some hydrophilic polymers, namely that associated with the lower critical solubility temperature (LCST). The polymers used in this study were poly[bis‐(2‐(2‐methoxyethoxy)ethoxy)phosphazene], MEEP (1), and two new water‐soluble polymers. These polymers are similar to MEEP, yet they contain a small percentage of a crosslinkable pendant group; either 2‐hydroxyethyl allyl ether (2), or o‐allyl phenol (3). The observed behavior was quite different for these two polymers than that found for MEEP, and is a direct consequence of the pendant group substitution patterns. Although the homopolymer MEEP yielded a single sharp LCST point, the two heteropolymers exhibited this phase transition over a broader temperature range. Further, fractionation of polymer 3, based on pendant group speciation, was possible due to the more hydrophobic nature of the phenol. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 78: 1092–1099, 2000     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

A single Fc binding domain-alkaline phosphatase gene fusion expresses a protein with both IgG binding ability and alkaline phosphatase enzymatic activity

A recombinant gene fusion was created and cloned using a previouslyconstructed gene encoding a monodomain IgG Fc binding proteinand the gene coding for bacterial alkaline phosphatase. Theconstruct was able to express and secrete a fusion protein thatexhibited both IgG binding and alkaline phosphatase enzymaticactivities. Greater than 60% of the protein demonstrating bothbiological activities was detected from periplasmic space preparations.Nanogram concentrations of the Fc binding-alkaline phosphatasefusion protein allowed primary IgG antibody detection withoutthe use of conjugated secondary antibodies. Removal of the domaincoding for alkaline phosphatase resulted in decreased resistanceof the protein to proteolytic degradation and the loss of IgGFc binding ability. Using affinity-purified fusion protein,the specificity of binding to IgG, IgM and IgA was examined;binding was strong to IgG and barely detectable against IgMor IgA. Affinity for binding of the fusion protein to IgG (k_d= 6.7 x10^-8 M) was determined to be equal to or greater thanpreviously reported for protein A.     

A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein

An Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 (isolate Wuhan-Hu-1) spike protein would significantly accelerate the search for anti-COVID-19 therapeutics because of its versatility and low cost. However, RBD contains four disulfide bonds and its expression in E. coli is limited by the formation of aberrant disulfide bonds resulting in inclusion bodies. Here, we show that a solubility-enhancing peptide (SEP) tag containing nine arginine residues (RBD-C9R) attached at the C-terminus can overcome this problem. The SEP-tag increased the expression in the soluble fraction and the final yield by five times (2 mg/L). The folding properties of the E. coli expressed RBD-C9R were demonstrated with biophysical characterization using RP-HPLC, circular dichroism, thermal denaturation, fluorescence, and light scattering. A quartz crystal microbalance (QCM) analysis confirmed the binding activity of RBD-C9R with ACE2, the host cell’s receptor. In addition, RBD-C9R elicited a Th-2 immune response with a high IgG titer in Jcl: ICR mice. The RBD-C9R antisera interacted with both itself and the mammalian-cell expressed spike protein (S1), as demonstrated by ELISA, indicating that the E. coli expressed RBD-C9R harbors native-like epitopes. Overall, these results emphasize the potential of our SEP-tag for the E. coli production of active multi-disulfide-bonded RBD.     

实验设计法在优化表达Fc融合蛋白的CHO细胞发酵培养工艺中的应用

目的通过实验设计(Design of Experiments,DOE)分析优化重组CHO细胞在生物反应器中的培养工艺。方法以2 L生物反应器控制参数温度(T)、pH及溶氧(dissolved oxygen,DO)作为变量,以蛋白表达量、蛋白纯度及蛋白唾液酸含量为响应变量,应用Design Expert 10软件进行3因素2水平DOE拟合分析,筛选最适表达Fc融合蛋白的CHO细胞发酵培养工艺参数,并采用5 L生物反应器进行验证。结果蛋白表达量仅与T有关,且呈成正相关,pH和DO对其无显著影响,T与pH对其有交互作用;蛋白纯度受T和pH影响显著,T呈负相关,pH呈正相关,DO对其无显著影响,T与pH对其有交互作用;唾液酸含量受T和pH影响显著,T呈正相关,pH呈负相关,DO对其无显著影响。最适工艺参数确定T为(34±0.5)℃,pH为(7.05±0.02),DO为(45±5)%。采用最适工艺参数的反应器与优化前工艺比较,活细胞密度(viable cell density,VCD)增加;蛋白表达量、蛋白纯度、唾液酸含量分别提高了29.7%、8%和87.9%。结论通过DOE方法快速优化了生物反应器培养表达Fc融合蛋白的CHO细胞的工艺参数,在不降低蛋白产量及纯度的前提下,极大提高了融合蛋白唾液酸含量。