衣藻叶绿体表达重组蛋白及表达优化策略

近年来,转基因技术已日趋成熟,医学、工业上的应用也越来越广泛。以重组蛋白为基础的药物治疗是目前医药生产领域发展最快的一项技术。它们的高特异性和低副作用使得治疗效率十分突出。但是重组蛋白表达的复杂性也给生产带来了一定限制。为了促进重组蛋白的应用,人们对适宜其表达的系统和能促进其表达的策略进行了探索。研究发现,衣藻叶绿体作为重组蛋白的生物反应器,能实现重组蛋白快速、高效、低成本生产。同时,衣藻能在人工培养基和人为控制的条件下生长,降低了受污染的风险,与传统的生产系统比较具有不可比拟的优越性。因此,衣藻叶绿体作为医药重组蛋白生物反应器在未来的生物技术领域将发挥巨大作用。     

衣藻叶绿体表达系统的研究

真核藻类作为一种新型的蛋白表达系统,因其培养方法简单,成本低廉并且能大规模繁殖,最近几年成为人们关注的焦点.作为一种模式生物,单细胞的真核生物莱茵衣藻已经成为人们研究的重点.外源蛋白不仅能在衣藻核中进行表达而且也能在叶绿体中表达,但衣藻叶绿体的表达系统较之核表达有巨大的优越性.在到目前为止,已经有许多的药用蛋白在莱茵衣藻的叶绿体中成功表达的报道,证明了莱茵衣藻叶绿体作为生物反应器的能力.将对衣藻叶绿体的表达做详细的描述.     

植物生物反应器研究现状、瓶颈及策略

近10年,植物作为重组蛋白生产系统是生命科学中研究最活跃领域之一。植物系统具有低成本、安全和易规模化优势,其表达生物活性药用蛋白能力已被许多研究所证实;同时,植物药用蛋白产品还表现出潜在的市场和广阔应用前景。鉴于此,回顾了植物生物反应器兴起,介绍了植物表达系统和重组蛋白研究现状,综述了植物生物反应器面临瓶颈问题、解决对策和未来一段时间内研究热点;在展望植物生物反应器前景同时,对我国研究现状、与国外差距和未来发展应采取策略进行了讨论。     

绿色荧光蛋白及其应用

绿色荧光蛋白是在水母中发现的新型报告分子,能在多种生物体内表达并发出荧光。对GFP中一些特定氨基酸进行突变可以产生多种类型的突变体,有利于研究蛋白之间或细胞器之间的相互作用。目前,GFP已经用于基因表达的报告、细胞动态的研究、活细胞内蛋白的定位及westernbloting检测中。GFP美好的应用前景也促进了有关GFP的研究,特别是寻找新的突变体并将之运用到细胞生物学和分子生物学的各个领域。     

利用植物生物反应器生产药用蛋白的研发现状

<正>植物生物反应器具有成本低、安全性高等优点,且植物具有真核细胞表达体系,能进行准确的蛋白修饰,使产品的免疫原性及生物活性较高,因此,植物生物反应器应用日益广泛。文章就现阶段植物生物反应器生产药用蛋白的研发及应用现状进行了综述,分析了目前本领域存在的主要技术瓶颈问题,并对利用植物生物反应器生产药用蛋白的发展前景进行了展望。     

利用动物乳腺生物反应器生产药用蛋白

动物乳腺生物反应器是利用动物乳腺特异性启动子调控元件指导外源基因在乳腺中特异性表达,并从转基因动物奶液中获取重组蛋白。应用动物乳腺生物反应器生产药用蛋白具有生产方式简单,产量大,蛋白能进行翻译后修饰等优点,是具有广阔前景的生物医药产业。本文仅就动物乳腺生物反应器的建立、检测、目的蛋白的分离纯化以及存在的问题等作一综述。     

猪瘟表面抗原E2-Erns蛋白在亚麻芥种子中的表达及活性分析

猪瘟病毒能导致猪的高度接触性传染病,对养猪业危害极大。由于传统猪瘟疫苗在使用及生产中存在诸多不足,促使科研工作者研制开发新型猪瘟疫苗。猪瘟病毒主要保护性抗原是E2蛋白和Erns蛋白,能够引起机体的免疫反应。植物表达体系具有表达效果好和生产成本低等优点,随着近些年开始出现利用转基因植物生产蛋白产品的技术,植物生物反应器越来越引起广泛关注。植物生物反应器具有完整的真核细胞表达系统,能够进行准确的蛋白翻译后加工,能较好的保留蛋白生物活性。为利用转基因植物生物反应器制备猪瘟的亚单位可饲疫苗及其商品化提供实验依据,本研究拟构建种子特异性启动子驱动的目的基因的表达载体,并转导至亚麻芥生物反应器中,表达猪瘟病毒E2-Erns融合蛋白。利用表达的猪瘟病毒E2-Erns蛋白以口服接种的方式进行小鼠模型的免疫实验,验证目标蛋白的抗原性。     

荧光蛋白在肿瘤分子影像研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）是在水母中发现的新型报告分子,该蛋白及其衍生物能在多种生物体内表达并发出荧光,是目前在生物学中研究和开发应用得最广泛的蛋白质之一。尤其是在肿瘤的研究中,荧光蛋白的分子影像可为深入揭示肿瘤发生发展的病理过程的机制,以及对其治疗进行实时、动态、细致、无创、靶向性的探测和跟踪提供有效手段。     

sTRAIL基因的表达、纯化及其生物学活性的初步研究

在原核表达系统中表达重组人可溶性肿瘤坏死因子相关的凋亡诱导配体(sTRAIL)分子,纯化表达产物,并进行初步的生物学活性的研究中,通过提取外周血淋巴细胞总RNA进行RT—PCR克隆sTRAIL cDNA,构建sTRAIL的原核表达载体,用限制性酶切和DNA测序进行鉴定,IPTG诱导表达。以发酵罐放大培养,SDS-PAGE和Western-blot确认表达,用亲和层析和阳离子层析纯化表达产物,使用L929、Qay肝癌、K562人白血病等肿瘤细胞鉴定活性。结果发现重组表达的sTRAIL融合蛋白占总蛋白的39％,单纯蛋白纯化后纯度达95％,用抗人TRAIL多抗可以确认表达了TRAIL分子,能诱导几株肿瘤细胞凋亡。原核表达系统正确表达了TRAIL分子,并摸索了中试生产的条件,为其在肿瘤生物治疗中的应用奠定了基础。     

乳腺生物反应器的表达优化策略

乳腺生物反应器是指将外源基因导入动物基因组并在动物乳腺中特异性表达,利用动物乳腺合成、分泌蛋白的功能,在其乳汁中获得外源蛋白的技术。乳腺生物反应器凭借其高表达、低成本以及合成蛋白质的结构接近天然蛋白质等优势而被视为药用和营养蛋白生产的一次技术革新,然而由于外源基因随机整合以及重组蛋白表达不稳定等问题极大地限制了其应用。本文结合乳腺生物反应器的发展现状,从利用基因编辑技术、筛选合适的外源基因整合位点以及改进外源基因调控序列3个方面对乳腺生物反应器优化策略进行了综述,以期为提高乳腺生物反应器生产重组蛋白的表达提供理论借鉴。     

Protein structure prediction using mutually orthogonal Latin squares and a genetic algorithm

We combine a new, extremely fast technique to generate a library of low energy structures of an oligopeptide (by using mutually orthogonal Latin squares to sample its conformational space) with a genetic algorithm to predict protein structures. The protein sequence is divided into oligopeptides, and a structure library is generated for each. These libraries are used in a newly defined mutation operator that, together with variation, crossover, and diversity operators, is used in a modified genetic algorithm to make the prediction. Application to five small proteins has yielded near native structures.     

Ribosome display selection of a metal-binding motif from an artificial peptide library

A new ribosome display system was applied for the in vitro selection of a metal-binding motif from an artificial peptide library. The display system consisted of an mRNA-associating protein, a ribosome, and mRNA. The protein part of this display system was designed to provide a random peptide library and to stabilize the ribosome display. The random peptide library was newly designed to isolate stable metal-binding motifs. We employed the system for in vitro selection and found several new proteins and peptides that bind Co(II)-immobilized resin and Co(II)-complex, respectively. This newly developed system can be conveniently applied to the in vitro selection of peptide aptamers.     

Glycosylation site binding protein, a component of oligosaccharyl transferase, is highly similar to three other 57 kd luminal proteins of the ER

A 57 kd component of oligosaccharyl transferase, termed glycosylation site binding protein, specifically recognizes a photoaffinity probe containing the N-glycosylation site sequence Asn-Lys-Thr. It is present in the lumen of the ER (endoplasmic reticulum) and its release from this compartment results in a loss of N-glycosylation. Antibodies against this protein were used to identify cDNA clones from a lambda gt11 expression library. Analysis of its cDNA sequence reveals high sequence similarity to three other 57 kd luminal endoplasmic reticulum proteins: protein disulfide isomerase, the beta-subunit of prolyl hydroxylase, and thyroid hormone binding protein. This finding suggests that the capacity to recognize multiple polypeptide domains may reside in a single luminal protein that participates in co- and/or posttranslational modifications of newly synthesized proteins.     

Enzymatic profiling of human kallikrein 14 using phage-display substrate technology

The human KLK14 gene is one of the newly identified serine protease genes belonging to the human kallikrein family, which contains 15 members. KLK14 , like all other members of the human kallikrein family, is predicted to encode for a secreted serine protease already found in various biological fluids. This new kallikrein is mainly expressed in prostate and endocrine tissues, but its function is still unknown. Recent studies have demonstrated that KLK14 gene expression is up-regulated in prostate and breast cancer tissues, and that higher expression levels correlate with more aggressive tumors. In this work, we used phage-display substrate technology to study the substrate specificity of hK14. A phage-displayed random pentapeptide library with exhaustive diversity was screened with purified recombinant hK14. Highly specific and sensitive substrates were selected from the library. We show that hK14 has dual activity, trypsin- and chymotrypsin-like, with a preference for cleavage after arginine residues. A SwissProt database search with selected sequences identified six potential human protein substrates for hK14. Two of them, laminin alpha-5 and collagen IV, which are major components of the extracellular matrix, have been demonstrated to be hydrolyzed efficiently by hK14.     

A novel rat orthologue and homologue for the Drosophila crooked neck gene in neural stem cells and their immediate descendants

The crooked neck (crn) gene of Drosophila melanogaster encodes a scaffold protein carrying multiple tetratricopeptide repeat (TPR) motifs, and its mutation results in a reduction in the number of neuroblasts and lethality during larval stages. Here, we isolated two structurally related genes from a rat embryonic brain cDNA library. One gene is the rat orthologue of crn, which encodes 690 amino acids including 16 copies of TPR. The other gene, ATH55, encodes an 855 amino acid protein including 21 TPR motifs, which presumably represents a rat crn homologue and an orthologue of human XAB2. Both genes are highly expressed in embryonic brain but their expressions decrease during development. ATH55-like immunoreactivity is present in the ventricular zone and newly formed cortical plate, while CRN-like immunoreactivity is more abundant in a younger ventricular zone. In agreement, both proteins were found to be enriched in cultured neural stem cells and to decrease in response to cell differentiation signals. As indicated for the yeast CRN-like protein, ATH55 and CRN immunoreactivities were both recovered in the nuclear fraction and detected in the splicing complex carrying pre-mRNA. These findings suggest that both TPR-motif-containing proteins are involved in RNA processing of mammalian neural stem cells and their immediate descendants.     

Molecular cloning of smg p21B and identification of smg p21 purified from bovine brain and human platelets as smg p21B

We have previously purified smg p21 from bovine brain membranes and isolated its cDNA from a bovine brain cDNA library. In the present studies, we have performed extensive screening of the bovine brain cDNA library with the cloned smg p21 cDNA as a probe and isolated another cDNA encoding a protein highly homologous to smg p21. The proteins encoded by the previously and newly isolated cDNAs are designated as smg p21A and -B, respectively. Since the partial amino acid sequences determined previously from the smg p21 purified from bovine brain were identical with the common amino acid sequences between smg p21A and -B, we have further sequenced smg p21 and identified it as smg p21B. We have also further sequenced the smg p21 purified from human platelet membranes and identified it as smg p21B. Amino acid sequence analysis indicates that smg p21A is identical with the rap1A and Krev-1 proteins and smg p21B is identical with the rap1B protein.     

选择感染性噬菌体技术及应用

选择感染性噬菌体技术是在传统噬菌体表面展示技术的基础上发展而来的一种新方法,通过将噬菌体感染力的恢复与蛋白间的相互作用偶联起来进行筛选,有效提高了筛选效率,简化了操作过程,降低了本底,为后基因组学的研究提供了便利方法,特别是体内法在理论上适合于库-库筛选,更具有应用价值。     

Evaluation of different glutathione S-transferase-tagged protein captures for screening E6/E6AP interaction inhibitors using AlphaScreen

Human papillomavirus (HPV) infection is responsible for the development of cervical cancer and its premalignant lesions in women. The virus-encoded oncogene E6 is a promising target for an anti-HPV drug therapy. The authors describe the development of a homogenous screening assay for inhibitors of the E6 interaction with its cellular target, the E6-associated protein (E6AP), based on AlphaScreen technology. The E6 protein was expressed and purified as glutathione S-transferase (GST) fusion protein, and the binding to a biotinylated E6AP peptide was monitored using GST-detecting Acceptor beads coated either with anti-GST antibody or glutathione. After optimization of the assay conditions, a commercial library of 3000 compounds was screened for inhibitors. Active compounds were retested and counterscreened for E6/E6AP specificity using biotinylated GST as a control protein. The results obtained with both types of GST-detecting reagents correlated very well and demonstrated the great potential of the newly developed glutathione-coated Acceptor beads as a detection reagent for GST fusion proteins.     

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize

A full-size cDNA clone (1614 bp) encoding calreticulin was isolated from a PCR-based cDNA library of maize in vitro zygotes. Calreticulin is a major Ca²⁺ storage protein located mainly in the lumen of the endoplasmic reticulum but also in the nucleus and/or cytoplasm of some cells. A differential screening between cDNA libraries originating from 104 in vitro zygotes (18 h after in vitro fertilization) and 128 unfertilized egg cells was performed to isolated newly expressed genes or genes expressed more abundantly after fertilization. The expression of the isolated cDNA clone is enhanced after fertilization and strongly correlated to cell division. Sequence comparison to a shorter maize calreticulin cDNA isolated from a conventional cDNA library proves the ability and reproducibility of the recently described method for PCR based cDNA library construction from a few plant cells [12]. It is further shown that calreticulins in maize are probably transcribed from a small gene family differentially expressed in abundance in diverse tissues. The deduced amino acid sequence encodes an acidic protein (pI 4.17) of 48 kDa sharing 77–92% and 50–54% homology to other plant and animal calreticulins, respectively. The described calreticulin gene represents to our knowledge the first cDNA clone isolated from a RT/PCR cDNA library originating from only a few plant cells and is the first gene isolated from zygotes of higher plants.