人血清白蛋白-睫状神经营养因子突变体融合蛋白基因在毕赤酵母中的表达及表达产物的纯化和活性鉴定

为了延长人睫状神经营养因子突变体的体内半衰期 ,将人血清白蛋白 (HAS)的C 末端和人睫状神经营养因子突变体AX15 (R13K)的N 末端通过一个 11个氨基酸的连接肽融合在一起 ,构建了融合蛋白HAS-AX15 (R13K)。HAS-AX15 (R13K)融合蛋白基因在巴斯德毕赤酵母中进行表达后通过阳离子交换层析、反向层析和凝胶过滤对表达产物进行了分离纯化。体外TF 1细胞存活实验表明与HAS融合并未影响AX15 (R13K)的生物学活性。体内动物实验表明HAS-AX15 (R13K)融合蛋白的疗效比AX15 (R13K)更为持久 :每 3天注射一次 4 8mg/kg的HAS-AX15 (R13K)融合蛋白的治疗效果优于每天注射一次 1 6mg/kg的AX15 (R13K)的治疗效果。HAS-AX15(R13K)融合蛋白不但可以减少用药次数 ,提高病人的顺应性 ,而且还可以减少用药量和血药浓度的波动 ,从而降低副反应 ,在临床应用上具有一定的优势。     

高比活人睫状神经营养因子突变体基因的构建及其在巴斯德毕赤酵母中的表达

人睫状神经营养因子(hCNTF)及其突变体有望成为治疗肥胖症的新型药物。为了减少hCNTF的副反应,提高其疗效,在hCNTF四重突变体AX15 (R13K)的基础上引入S16 5D Q16 6H突变,构建了高比活的DH_AX15 (R13K)突变体。体外和体内实验表明DH_AX15 (R13K)的活性约是AX15 (R13K)的5倍。同时体内实验还发现DH_AX15(R13K)的作用比AX15 (R13K)更为持久。这种更为持久的作用可能是由于活性提高而非半衰期延长引起的。高比活的hCNTF突变体一方面可以在保证疗效的前提下减少蛋白用量,减少副反应;另一方面可以在不增加副反应的前提下增加最大耐受剂量,提高疗效,在临床应用上具有潜在的优势     

人睫状神经营养因子结构和功能的研究

睫状神经营养国子在神经系统的发育和损伤修复过程中具有重要作用。本文根据由核苷酸序列推导的氨基酸序列预测了人睫状神经营养因子和二级结构。参考结构预测结果,用片段插入法和缺失地,改造人睫状神经营养因子编码基因,在大肠菌中表达并纯化了五系人睫状神经营养因子的突变体,观察结构改造对人睫状神经营养因子神经营养活性的影响。     

定量测定重组人睫状神经营养因子活性的新方法

目的：建立一种能定量测定重组人睫状神经营养因子（ciliary neurotrophic factor,CNTF）生物学活性的新方法。方法：从鸡胚中分离出背根神经节并制成神经细胞,将重组人睫状神经营养因子加入到细胞中继续培养64h后,用酸性磷酸酶法检测活细胞内酸性磷酸酶的活性,从而定量测定重组人睫状神经营养因子的生物活性。结果：重组人睫状神经营养因子有促原代鸡胚背根神经细胞存活作用,细胞存活率与加入重组人睫状神经营养因子的量成正相关。结论：通过检测存活的原代鸡胚背根神经细胞内酸性磷酸酶的含量来定量测定重组人睫状神经营养因子生物活性的实验方法具有干扰因素少、定量准确、重复性好等优点。     

睫状神经营养因子研究进展

睫状神经营养因子（ＣＮＴＦ）能够促进多种神经元的存活,在神经系统发育、分化和损伤修复过程中具有重要作用。睫状神经营养因子与白血病抑制因子、白细胞介素６有相似的空间结构,它们的受体组成也相关。睫状神经营养因子的神经营养作用研究为临床治疗神经系统疾病带来了新的希望。     

人睫状神经营养因子突变体基因克隆及高效表达

为了提高人睫状神经营养因子（ＣＮＴＦ）的生物学活性,用ＰＣＲ方法获取Ｎ端缺失１４个氨基酸的ＣＮＴＦ基因片段,经酶切鉴定、核酸测序证实突变体的核苷酸序列,将其重组至表达质粒ｐＢＶ２２０,构建了ＣＮＴＦ突变体表达载体ｐＢＶ－ＣＮＴＦΔ．用ＳＤＳ－ＰＡＧＥ测定其表达水平,鸡胚背根节无血清培养法检测表达蛋白的生物学活性．结果表明,ｐＢＶ－ＣＮＴＦΔ能表达生物学活性高于天然ＣＮＴＦ的约２６ｋＤ蛋白质,表达水平达３０％．为今后通过基因工程方法获得ＣＮＴＦ突变体,从而制备高效的ＣＮＴＦ制剂创造了条件．     

人睫状神经营养因子的基因克隆与高效表达

人睫状神经营养因子的基因克隆与高效表达范明,杜方勇,咸海清,刘淑红,甘思德（军事医学科学院基础医学研究所北京１００８５０）睫状神经营养因子（ｃｉｌｉａｒｙｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ＣＮＴＦ）以最早发现于鸡睫状神经节而命名,是目前已知的神经...     

神经再生过程中睫状神经营养因子基因表达的变化

神经再生过程中睫状神经营养因子基因表达的变化甘思德,杜方勇,范明（军事医学科学院基础医学研究所北京１００８５０）根据现有资料,在众多的神经因子（ｎｅｕｒｏｋｉｎｅｓ）中,睫状神经营养因子（ＣＮＴＦ）与神经营养素家族（ｎｅｕｒｏｔｒｏｐｈｉｎｅｓ）完全...     

睫状神经营养因子的研究进展

睫状神经营养因子 (ＣｉｌｉａｒｙＮｅｕｒｏｔｒｏｐｈｉｃＦａｃ ｔｏｒ ,ＣＮＴＦ )最初是从鸡胚的眼组织睫状节中提取出来 ,因对睫状节神经元有营养作用并可维持鸡副交感神经节的存活而得名[1～ 3] 。ＣＮＴＦ属神经调节细胞因子家族中的一员 ,但不属于神经营养因子家族成员。至今 ,已发现ＣＮＴＦ具有广泛的生物学活性 ,如它对于感觉和运动神经元的分化、存活及功能维持均具有重要作用[1,4 ] 。本文就ＣＮＴＦ的结构、分布、生物学效应及其与脊髓损伤修复的关系作一综述。1.ＣＮＴＦ及其基因结构1 1　ＣＮＴＦ的结构ＣＮＴＦ最初由Ｈ…     

睫状神经营养因子突变体蛋白的活性研究

为了进一步研究我室应用计算机分子模拟设计并表达纯化的睫状神经营养因子突变体蛋白的生物学活性,分别采用鸡胚背根神经节无血清培养法、TF-1细胞增殖法、正常小鼠减重法对其活性进行研究。结果是突变体蛋白能促进鸡胚背根神经节的生长;促进TF-1细胞增殖,MTT测定法表明突变体蛋白与国际参考品相比,比活不低于2.0×106U/mg;使正常小鼠的体重减轻,摄食量减少,脂肪指数下降,并且体重的减轻与突变体蛋白的给药剂量呈现良好的剂量依赖关系,其ED50为：150.986?g/kg/d。以上实验表明CNTF突变体蛋白具有促神经生长、促TF-1细胞增殖和减重的生物学活性。从而为其进一步的应用和开发提供了线索。     

T cells redirected to interleukin-13Rα2 with interleukin-13 mutein–chimeric antigen receptors have anti-glioma activity but also recognize interleukin-13Rα1

Background aimsOutcomes for patients with glioblastoma remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)13Rα2, human epidermal growth factor receptor 2, epidermal growth factor variant III or erythropoietin-producing hepatocellular carcinoma A2 has shown promise for the treatment of glioma in preclinical models. On the basis of IL13Rα2 immunotoxins that contain IL13 molecules with one or two amino acid substitutions (IL13 muteins) to confer specificity to IL13Rα2, investigators have constructed CARS with IL13 muteins as antigen-binding domains. Whereas the specificity of IL13 muteins in the context of immunotoxins is well characterized, limited information is available for CAR T cells.MethodsWe constructed four second-generation CARs with IL13 muteins with one or two amino acid substitutions, and evaluated the effector function of IL13-mutein CAR T cells in vitro and in vivo.ResultsT cells expressing all four CARs recognized IL13Rα1 or IL13Rα2 recombinant protein in contrast to control protein (IL4R) as judged by interferon-γ production. IL13 protein produced significantly more IL2, indicating that IL13 mutein–CAR T cells have a higher affinity to IL13Rα2 than to IL13Rα1. In cytotoxicity assays, CAR T cells killed IL13Rα1- and/or IL13Rα2-positive cells in contrast to IL13Rα1- and IL13Rα2-negative controls. Although we observed no significant differences between IL13 mutein–CAR T cells in vitro, only T cells expressing IL13 mutein–CARs with an E13K amino acid substitution had anti-tumor activity in vivo that resulted in a survival advantage of treated animals.ConclusionsOur study highlights that the specificity/avidity of ligands is context-dependent and that evaluating CAR T cells in preclinical animal model is critical to assess their potential benefit.     

Yeast KEX2 genes encodes an endopeptidase homologous to subtilisin-like serine proteases

Yeast Saccharomyces cerevisiae KEX2 gene previously isolated, was characterized as the gene encoding a calcium-dependent endopeptidase required for processing of precursors of alpha-factor and killer toxin. In this study, we report the amino acid sequence of the KEX2 gene product deduced from nucleotide sequencing. Our results indicate that the KEX2 gene contains a 2,442-bp open reading frame encoding a polypeptide of 814 amino acids. The deduced amino acid sequence contains a region extensively homologous to the members of subtilisin-like serine protease family near the N-terminus. A putative membrane-spanning domain near the C-terminus was also detected. These facts indicate that the KEX2-encoded protein may function as a membrane-bound, subtilisin-like serine protease.     

Variation among lines selected for body size in the fractional rate of degradation of protein and acid protease activity in the muscle of quail (Coturnix coturnix japonica)

Fractional rates (%/day) of degradation of muscle protein were determined by measuring the output of NT-methylhistidine (NT-MH) in the excreta at 2 and 10 weeks of age in three lines of quail, a random-bred line and two lines selected for body size, one for increased and the other for decreased size. In all lines, fractional rates of degradation of muscle protein at 2 weeks of age were higher than those at 10 weeks of age. The fractional rate of degradation at 2 weeks of age was highest for the RR line, 9.1-9.2%/day. However, at 10 weeks of age, the rank order changed, and the RR line showed the lowest rate, 1.8-1.9%/day. The SS line (5.8-6.2%/day at 2 weeks and 5.8-5.9%/day at 10 weeks of age) was significantly higher than the LL line (4.1-4.2%/day at 2 weeks and 2.1-2.2%/day at 10 weeks of age). Acid protease activities in supernatants of homogenized muscle of the three lines of quail at 2 and 10 weeks of age were measured. In all lines, the acid protease activities in supernatant of homogenized muscle decreased from 2 to 10 weeks of age. At 2 weeks, the protease activity of the RR line was significantly higher than that of the LL and SS lines, which did not differ significantly. However, at 10 weeks of age, the SS line had higher activity in both sexes than the LL and RR lines. The results suggest that selection for body size brings about significant changes in both fractional degradation rate and acid protease activity in the muscle.     

Evaluation of peptide-aldehyde inhibitors using R188I mutant of SARS 3CL protease as a proteolysis-resistant mutant

The 3C-like (3CL) protease of the severe acute respiratory syndrome (SARS) coronavirus is a key enzyme for the virus maturation. We found for the first time that the mature SARS 3CL protease is subject to degradation at 188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis. The R188I mutant digested a conserved undecapeptide substrate with a K(m) of 33.8 microM and k(cat) of 4753 s(-1). Compared with the value reported for the mature protease containing a C-terminal His-tag, the relative activity of the mutant was nearly 10(6). Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently inhibited the catalytic activity of the R188I mutant. The results indicated for the first time that the tetrapeptide sequence is enough for inhibitory activities of peptide-aldehyde derivatives.     

Degradation of HSA-AX15(R13K) when expressed in Pichia pastoris can be reduced via the disruption of YPS1 gene in this yeast

Expression of recombinant protein HSA-AX15(R13K) in Pichia pastoris GS115 strain produced both the intact protein and its two degradation products with molecular weights of around 43kDa and 66.2kDa, respectively. To reduce or avoid the degradation, a modified P. pastoris GS115 stain, in which YPS1 gene was disrupted, was constructed via homologous recombination and used as a host strain for the HSA-AX15(R13K) expression. After 60h of induction during culture, it was found that the degradation product of around 66.2kDa was reduced significantly in the supernatant of yps1-disrupted strain compared with that in the supernatant of wild-type strain. By the Western blot analysis of culture supernatants from wild-type and yps1-disrupted strains expressing HSA-AX15(R13K), the significant improvement was also seen in the degradation product of around 43kDa. Comparison of cell growth between the two strains demonstrated a similar growth tendency, thereby indicating that the disruption of YPS1 gene has no effect on the normal physiology of GS115 strain. Following induction for 60h, the yield of intact HSA-AX15(R13K) in the yps1 disruptant was three-fold higher than that in the wild-type strain. Therefore, such a P. pastoris mutant deficient in YPS1 activity is suitable for the high-level expression of recombinant protein HSA-AX15(R13K).     

Proteasomes cleave at multiple sites within polyglutamine tracts: activation by PA28gamma(K188E)

Eukaryotic proteasomes have been reported to cleave only once within polyglutamine tracts and then only after the N-terminal glutamine (Venkatraman, P., Wetzel, R., Tanaka, M., Nukina, N., and Goldberg, A. L. (2004) Mol. Cell 14, 95-104). We have obtained results that directly conflict with that report. In the presence of the proteasome activator PA28gamma(K188E) human red cell proteasomes progressively degraded fluorescein-GGQ(10)RR or fluorescein-HPHQ(10)RR into small fragments as shown by size exclusion chromatography and mass spectrometry. MALDI-TOF mass spectrometry revealed that proteolytic products arose from cleavage after every glutamine in fluorescein-HPHQ(10)RR, and mass accuracy rules out deamidation of glutamine to glutamic acid as an explanation for peptide degradation. Moreover, degradation cannot be attributed to a contaminating protease because peptide hydrolysis was completely blocked by the proteasome-specific inhibitors, lactacystin and epoxomicin. We conclude that proteasomes cleave repetitively anywhere within a stretch of ten glutamine residues. Thus our results cast doubt on the idea that mammalian proteasomes cannot degrade glutamine-expanded regions within pathogenic polyQ-expanded proteins, such as Huntingtin.     

Blockade of the sympathetic nervous system degrades ligament in a rat MCL model.

We hypothesize that blockade of the sympathetic nervous system degrades ligament. We tested this hypothesis in a rat medial collateral ligament (MCL) model. Fifteen animals were treated for 10 days with the sympathetic chemotoxin guanethidine using osmotic pumps, whereas 15 control rats received pumps containing saline. A reduction in plasma concentrations of norepinephrine in the guanethidine rats indicated a significant decrease in sympathetic nerve activity. Vasoactive intestinal peptide and neuropeptide Y were decreased in MCLs from guanethidine animals, as quantified by radioimmunoassays. Tissue vascularity was substantially increased in guanethidine MCLs, whereas mechanical properties were significantly decreased. Proteases, such as matrix metalloproteinases (MMP) and cysteine proteases, play a major role in ligament degradation. The proteases MMP-13, cathepsin K, and tartrate-resistant acid phosphatase (TRAP) have collagenolytic activity and have been shown in rat ligament tissues. To determine whether the degradation seen in this study was due to protease activity, we determined the expression of these enzymes in control and treated MCLs. Real-time quantitative PCR revealed that guanethidine treatment increased expression of MMP-13 and cathepsin K mRNAs, although overall expression levels of MMP-13 and TRAP were relatively low. Histology also identified increases in TRAP and cathepsin K, but not MMP-13, in guanethidine-treated tissues. Results support our hypothesis that blockade of the sympathetic nervous system substantially degrades ligament.     

Engineering soluble monomeric streptavidin with reversible biotin binding capability

Monomeric streptavidin with reversible biotin binding capability has many potential applications. Because a complete biotin binding site in each streptavidin subunit requires the contribution of tryptophan 120 from a neighboring subunit, monomerization of the natural tetrameric streptavidin can generate streptavidin with reduced biotin binding affinity. Three residues, valine 55, threonine 76, and valine 125, were changed to either arginine or threonine to create electrostatic repulsion and steric hindrance at the interfaces. The double mutation (T76R,V125R) was highly effective to monomerize streptavidin. Because interfacial hydrophobic residues are exposed to solvent once tetrameric streptavidin is converted to the monomeric state, a quadruple mutein (T76R,V125R,V55T,L109T) was developed. The first two mutations are for monomerization, whereas the last two mutations aim to improve hydrophilicity at the interface to minimize aggregation. Monomerization was confirmed by four different approaches including gel filtration, dynamic light scattering, sensitivity to proteinase K, and chemical cross-linking. The quadruple mutein remained in the monomeric state at a concentration greater than 2 mg/ml. Its kinetic parameters for interaction with biotin suggest excellent reversible biotin binding capability, which enables the mutein to be easily purified on the biotin-agarose matrix. Another mutein (D61A,W120K) was developed based on two mutations that have been shown to be effective in monomerizing avidin. This streptavidin mutein was oligomeric in nature. This illustrates the importance in selecting the appropriate residues and approaches for effective monomerization of streptavidin.