集成干扰素突变体Ⅱ的分子构建、表达及提纯

目的：通过定点突变,构建集成干扰素突变体Ⅱ（IFN-Con-m2）,以期获得兼具高效作用和可定点聚乙二醇（PEG）修饰的新型药物分子。 方法：采用PCR体外定点突变技术,使集成干扰素突变体Ⅰ（IFN-Con-m1）基因的第86位密码子由TAC突变为TGC。将扩增片段克隆入pET-23b表达载体,重组质粒转化大肠杆菌BL21（DE3）。IPTG诱导后,表达的IFN-Con-m2经包含体变复性、疏水层析、DEAE层析和凝胶过滤层析等纯化后,用WISH-VSV系统进行生物活性测定。 结果：IFN-Con-m2以包涵体形式表达,表达量占菌体总蛋白的30%以上。纯化后,IFN-Con-m2的纯度大于95%,比活性大于5.0×108IU/mg。 结论：构建了IFN-Con-m2的表达载体,并成功地在大肠杆菌中表达,获得了高活性突变分子IFN-Con-m2,建立了IFN-Con-m2的纯化工艺。     

噬菌体显示技术改造人干扰素α1c/86D的研究

利用噬菌体表面显示技术表达具生物活性的干扰素α1c,并通过构建和筛选突变文库 ,以得到高受体结合能力和高比活性的新α1c干扰素 .利用噬菌粒载体pCANTAB5E表达了人IFNα1c/ 86D基因 ,利用随机 6肽库和干扰素的中和抗体推导干扰素α1c的受体结合区及关键性结合残基 ,运用盒式突变构建了基于AB环的突变库 ,其中氨基酸 2 9,3 1,3 2 ,3 5位完全随机化 ,并直接利用WISH细胞选择出高抗病毒活性的干扰素突变体 .结果获得了 3个重组噬菌体干扰素突变体 ,其抗病毒活性是干扰素母体的 4～ 16倍 .这一结果为国内外首次报道利用噬菌体表面显示技术的筛选表达优势改造干扰素以提高抗病毒活性 ,实验结果说明了其可行性 ,并提示干扰素突变体的比活性有可能较母体高 .     

Gln49-磷脂酶A2基因工程定点突变及酶活性分析

为了确认49位谷氨酰胺磷脂酶A2（Glutamine 49 phospholipase A2, Gln49-PLA2）酶活性缺失与氨基酸序列的相关性,对Gln49-PLA2编码基因第49位氨基酸进行PCR定点突变,利用pET32a+质粒载体在大肠杆菌中表达Gln49-磷脂酶A2的突变体--天冬氨酸磷脂酶A2（Aspartic acid 49 phospholipase A2, Asp49-PLA2--Q49D-PLA2）。将表达的包涵体蛋白变性,采用固定化金属离子亲和层析进行柱上复性、纯化获得突变体融合蛋白（fusion Q49D-PLA2--fQ49D-PLA2）;突变体融合蛋白经蛋白水解酶Factor Xa酶切后,采用Hitrap SP阳离子交换层析和Superdex 75凝胶层析进一步纯化,得到突变体蛋白Q49D-PLA2,得率为1.3%,比酶活为72U/mg。从而证实Gln49-PLA2酶活性缺失的关键原因是49位氨基酸为谷氨酰胺。     

大肠杆菌磷酸甘油酸脱氢酶突变体的构建及抗反馈抑制效应

磷酸甘油酸脱氢酶(D-3-phosphoglycerate dehydrogenase,PGDH,EC 1.1.1.95)为L-丝氨酸合成途径的关键酶,其编码基因为ser A,其活性受到合成产物L-丝氨酸的反馈抑制调控。为解除丝氨酸的反馈抑制,采用定点突变技术把编码PGDH酶344位组氨酸或346位天冬氨酸或364位天冬氨酸的密码子定点突变为丙氨酸密码子。改造后的ser AFbr被连到表达载体pT7-7上,并转入大肠杆菌Escherichia coli BL21(DE3)中进行表达,破壁回收粗酶液,通过DEAE阴离子柱纯化PGDH突变体,并对其酶活性和IC_(50)值进行了测定。结果,野生型PGDH酶IC_(50)值为7μmol/L,而PGDH双突变体N346A/H344A催化活性与野生型相近,在丝氨酸浓度为160 mmol/L时,其酶活仍保持未添加丝氨酸时酶活的96%,基本解除反馈抑制。     

集成干扰素突变体IIFN/165S的构建及其生物学评价

目的:通过定点突变,构建集成干扰素(IIFN/165S),以期获得高效的新型药物分子.方法:采用PCR体外定点突变技术,使集成干扰素IIFN基因的第165位密码子由CGT突变为AGT.扩增片段克隆入pET-23b表达载体,重组质粒转化大肠杆菌BL21(DE3).在LB培养基中培养,经IPTG诱导表达的IIFN/165S经包涵体变性、复性以及层析纯化后,经SDS-PAGE、Western blot和MALDI-TOF-MS分析,用WISH-VSV系统进行抗病毒活性测定同时应用流式细胞术检测细胞凋亡率.结果:IIFN/165S以包涵体形式表达.纯化后,IIFN/165S的纯度大于95％,分子量为18172,比活性(7.63±0.22)×108 IU/mg,诱导细胞凋亡率呈剂量依赖.结论:构建了IIFN/165S的表达载体,并成功地在大肠杆菌中表达,获得了高纯度高活性突变分子IIFN/165S.     

重组猪a干扰素基因定点突变及在大肠杆菌中的表达

用巨引物PCR法介导的定点突变把猪α干扰素(PoIFN-α)第86位Cys(TGC)突变为Tyr(TAC),同时将其成熟蛋白N端第一个密码子TGT同义突变大肠杆菌偏爱的密码子TGC,构建了大肠杆菌融合基因表达载体pGEX-IFN,表达产物占菌体总蛋白的20%.将以包涵体形式表达的目的蛋白经变、复性处理,并以FPLC进一步纯化,得到了具有较高生物活性的产物(5200IU/mg).     

人干扰素α1b突变体IFNα1b/31K的构建及其生物学评价

目的:构建干扰素α1b突变体IFNα1b/31K,以期获得高效低毒的新型药物分子。方法:根据合理药物设计,采用定点突变技术,将干扰素α1b第31位氨基酸残基突变为K,并构建表达IFNα1b/31K重组蛋白。纯化后,对其抗病毒活性、抗肿瘤细胞增殖活性和动物体内急性毒性进行考察。结果:IFNα1b/31K表达量占菌体总蛋白的30%以上。纯化后的IFNα1b/31K纯度大于95%,比活性约为IFNα1b的1.7倍,抗肿瘤增殖活性比IFNα1b降低,未见对实验动物的急性毒性作用。结论:成功设计构建并表达了高效低毒的IFNα1b突变蛋白分子。     

重组猪α干扰素基因定点突变及在大肠杆菌中的表达

用巨引物PCR法介导的定点突变把猪α干扰素（PoIFNα）第86位Cys（TGC）突变为Tyr（TAC）,同时将其成熟蛋白N端第一个密码子TGT同义突变大肠杆菌偏爱的密码子TGC,构建了大肠杆菌融合基因表达载体pGEXIFN,表达产物占菌体总蛋白的20%。将以包涵体形式表达的目的蛋白经变、复性处理,并以FPLC进一步纯化,得到了具有较高生物活性的产物(5200IU/mg)。     

重组肿瘤坏死因子-α衍生物的制备及聚乙二醇修饰

应用搭桥PCR技术,将肿瘤坏死因子-α基因的前4位氨基酸的编码序列删除,对hTNF-α的第8/9/10/29/31/157位氨基酸的密码子进行定点突变,将突变后的cDNA插入到pBV220载体中构建重组质粒pBV220-tnf-αD4。将重组质粒转化大肠杆菌DH5α,筛选获得了高效表达TNF-αD4突变体的工程菌,表达的重组蛋白约占菌体蛋白总量的45%左右,经硫酸铵沉淀和阳离子交换层析纯化得到纯度达90%以上的重组目的蛋白,比活性达到8×107。用单甲氧基聚乙二醇-丁醛（mPEG-ButyrALD）对TNF-αD4进行修饰,经阳离子交换层析纯化得到纯的mPEG-TNF-αD4,纯度达85%以上,比活性达到8.6 ×107,系统毒性也有了明显的降低。此项工作通过应用PEG修饰肿瘤坏死因子-α,为降低其毒性,增加其活性进行了有益的尝试,为其进一步研究与开发奠定了基础。     

集成干扰素突变体IIFN72C的构建及其聚乙二醇定点修饰

目的：设计构建集成干扰素突变体IIFN72C并进行聚乙二醇定点修饰,以获得高活性的长效干扰素分子。方法：利用蛋白质分子同源模建,选择在集成干扰素分子IIFN的第72位引入半胱氨酸残基构成集成干扰素突变体IIFN72C。诱导表达后经包涵体变复性和层析纯化,与单甲氧基聚乙二醇（mPEGMAL）定点偶联。修饰产物经纯化后,以SDSPAGE考察其纯度,用WISHVSV系统进行生物活性测定。结果：IIFN72C以包涵体形式表达,表达量占菌体总蛋白的30%以上,比活性与突变前相当;修饰产物大多数为单修饰体,纯化后纯度大于98%,比活性保留约为修饰前的8%。结论：成功设计并表达IIFN72C用于PEG定点修饰,修饰产物活性保留得以提高。     

Role of His159 in yeast enolase catalysis.

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA. Metal binding studies support the above proposal. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281. Cha et al. (1994) J. Biol. Chem. 269, 2687-2694].     

新型集成干扰素-人血清白蛋白融合蛋白在毕赤酵母中的分泌表达和纯化

将含编码HSA天然信号肽、HSA和新型集成干扰素(NIFN)的融合基因,插入毕赤酵母表达载体pPIC3.5,转染毕赤酵母GS115,用于分泌表达融合蛋白HSA-NIFN的酵母工程菌。诱导表达后,产物经SDS-PAGE、Western blot和MALDI-TOF-MS分析表明该蛋白分子量为86369Da,且能被抗HSA单抗特异性结合;表达的HSA-NIFN经超滤、Blue亲和层析和CM阳离子交换层析纯化后,HSA-NIFN的纯度大于95%。用细胞病变抑制法测定其比活性为7.75±0.39×106IU/mg。     

His92 and His110 selectively affect different heme centers of adrenal cytochrome b(561)

Adrenal cytochrome b(561) (cyt b(561)), a transmembrane protein that shuttles reducing equivalents derived from ascorbate, has two heme centers with distinct spectroscopic signals and reactivity towards ascorbate. The His54/His122 and His88/His161 pairs furnish axial ligands for the hemes, but additional amino acid residues contributing to the heme centers have not been identified. A computational model of human cyt b(561) (Bashtovyy, D., Berczi, A., Asard, H., and Pali, T. (2003) Protoplasma 221, 31-40) predicts that His92 is near the His88/His161 heme and that His110 abuts the His54/His122 heme. We tested these predictions by analyzing the effects of mutations at His92 or His110 on the spectroscopic and functional properties. Wild type cytochrome and mutants with substitutions in other histidine residues or in Asn78 were used for comparison. The largest lineshape changes in the optical absorbance spectrum of the high-potential (b(H)) peak were seen with mutation of His92; the largest changes in the low-potential (b(L)) peak lineshape were observed with mutation of His110. In the EPR spectra, mutation of His92 shifted the position of the g=3.1 signal (b(H)) but not the g=3.7 signal (b(L)). In reductive titrations with ascorbate, mutations in His92 produced the largest increase in the midpoint for the b(H) transition; mutations in His110 produced the largest decreases in DeltaA(561) for the b(L) transition. These results indicate that His92 can be considered part of the b(H) heme center, and His110 part of the b(L) heme center, in adrenal cyt b(561).     

The distinct heme coordination environments and heme-binding stabilities of His39Ser and His39Cys mutants of cytochrome b5

A gene mutant library containing 16 designed mutated genes at His39 of cytochrome b(5) has been constructed by using gene random mutagenesis. Two variants of cytochrome b(5), His39Ser and His39Cys mutant proteins, have been obtained. Protein characterizations and reactions were performed showing that these two mutants have distinct heme coordination environments: ferric His39Ser mutant is a high-spin species whose heme is coordinated by proximal His63 and likely a water molecule in the distal pocket, while ferrous His39Ser mutant has a low-spin heme coordinated by His63 and Ser39; on the other hand, the ferric His39Cys mutant is a low-spin species with His63 and Cys39 acting as two axial ligands of the heme, the ferrous His39Cys mutant is at high-spin state with the only heme ligand of His63. These two mutants were also found to have quite lower heme-binding stabilities. The order of stabilities of ferric proteins is: wild-type cytochrome b(5) > His39Cys > His39Ser.     

Self-organized nanogels responding to tumor extracellular pH: pH-dependent drug release and in vitro cytotoxicity against MCF-7 cells

The principal objective of this study was to fabricate doxorubicin-loaded self-organized nanogels composed of hydrophobized pullulan (PUL)-Nalpha-Boc-L-histidine (bHis) conjugates. Their responses to tumor extracellular pH (pHe) were determined, and they were also evaluated with regard to their anticancer efficacy against breast cancer cell lines (MCF-7). bHis was grafted to a PUL-deoxycholic acid (DO) conjugate (PUL-DO) via an ester linkage. PUL-DO/bHis conjugates with two different degrees of bHis substitutions (PUL-DO/bHis36 and PUL-DO/bHis78) were synthesized. PUL-DO/bHis nanogels formed via dialysis at a pH of 8.5 evidenced larger particle sizes (<140 nm) and lower critical aggregation concentrations (CACs) than did the PUL-DO nanogels (90 nm). The pH-dependent CAC of PUL-DO/bHis78 changed dramatically, from 1.2 microg/mL at pH 8.5, to 10 at 7.0, and to 660 at 6.2. A similar tendency in pH-dependent size was also noted. The ionization of the imidazole ring in bHis is principally responsible for pH dependency. The bHis moieties function as a switching tool responding to external pH. Doxorubicin (DOX)-loaded nanogels were assessed for pH-dependent releasing kinetics. The release rate of DOX from the PUL-DO/bHis78 nanogels increased significantly with reductions in pH. This resulted in increased cytotoxicity (30% cell viability at a dose of 10 microg/mL DOX equivalent) against sensitive MCF-7 cells at a pH of 6.8 and low cytotoxicity at pH 7.4 (65% cell viability at an identical dose). The results show that PUL-DO/bHis nanogels may potentially be employed as anti-tumor drug carriers.     

His Bundle Activates Faster than Ventricular Myocardium during Prolonged Ventricular Fibrillation

Background

The Purkinje fiber system has recently been implicated as an important driver of the rapid activation rate during long duration ventricular fibrillation (VF>2 minutes). The goal of this study is to determine whether this activity propagates to or occurs in the proximal specialized conduction system during VF as well.

Methods and Results

An 8×8 array with 300 µm spaced electrodes was placed over the His bundles of isolated, perfused rabbit hearts (n = 12). Ventricular myocardial (VM) and His activations were differentiated by calculating Laplacian recordings from unipolar signals. Activation rates of the VM and His bundle were compared and the His bundle conduction velocity was measured during perfused VF followed by 8 minutes of unperfused VF. During perfused VF the average VM activation rate of 11.04 activations/sec was significantly higher than the His bundle activation rate of 6.88 activations/sec (p<0.05). However from 3–8 minutes of unperfused VF the His system activation rate (6.16, 5.53, 5.14, 5.22, 6.00, and 4.62 activations/sec significantly faster than the rate of the VM (4.67, 3.63, 2.94, 2.24, 3.45, and 2.31 activations/sec) (p<0.05). The conduction velocity of the His system immediately decreased to 94% of the sinus rate during perfused VF then gradually decreased to 67% of sinus rhythm conduction at 8 minutes of unperfused VF.

Conclusion

During prolonged VF the activation rate of the His bundle is faster than that of the VM. This suggests that the proximal conduction system, like the distal Purkinje system, may be an important driver during long duration VF and may be a target for interventional therapy.     

Challenging a paradigm: Theoretical calculations of the protonation state of the Cys25‐His159 catalytic diad in free papain

A central mechanistic paradigm of cysteine proteases is that the His–Cys catalytic diad forms an ion‐pair NH(+)/S(?) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X‐ray crystallography studies of viral and bacterial cysteine proteases depart from the ion‐pair mechanism, suggesting general base catalysis. We challenge the postulate of the ion‐pair formation in free papain. Applying our QM/SCRF(VS) molecular modeling approach, we analyzed all protonation states of the catalytic diad in free papain and its SMe derivative, comparing the predicted and experimental pK_a data. We conclude that the His–Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pK_a = 8.62 of His159 imidazole in free papain, obtained by NMR‐controlled titration and originally interpreted as the NH(+)/S(?) ? N/S(?) equilibrium, is now assigned to the NH(+)/SH ? N/SH equilibrium. Proteins 2009. © 2009 Wiley‐Liss, Inc.