信号肽对外源蛋白分泌效率的影响

信号肽在引导外源蛋白分泌过程中具有重要作用,从信号肽疏水性结构、构建分泌型载体以及分泌增强子、定位信号等几个方面介绍了信号肽对外源蛋白分泌效率的影响,在大量生产以酵母为表达栽体的治疗性蛋白方面具有广泛的应用前景。     

信号肽对外源蛋白分泌效率的影响

信号肽在引导外源蛋白分泌过程中具有重要作用,本文从信号肽疏水性结构、构建分泌型载体以及分泌增强子、定位信号等几个方面介绍了信号肽对外源蛋白分泌效率的影响,在大量生产以酵母为表达宿主的治疗性蛋白方面具有广泛的应用前景。     

大肠杆菌中的蛋白质分泌

目前,在微生物遗传工程中应用的表达载体大多是指导目的基因在宿主的胞质中表达,尽管外源蛋白的产量可达菌体总蛋白的50％以上,但同时也存在不少缺点:(1)高水平表达的外源蛋白容易发生沉降、凝聚,难以重新折叠为正确构象,不易获得有功能的产品;(2)产品纯化工艺复杂;(3)对宿主有毒性作用,导致表达体系不稳定;(4)外源蛋白易被胞内蛋白酶降解。这使得微生物遗传工程的应用受到很大限制。 如果能使在胞质表达的外源蛋白分泌到胞外,既可纠正由子高表达所引起的种种不利。近十年开展了对E.coli中蛋白质定位的深入研究。E.coli的蛋白质最初都是在胞质合成的,通过分泌而定位于膜或周质。例如外膜蛋白A(OmpA)先以前体形式在胞质合成,然后借助信号肽的作用穿过内膜,在分泌过程中信号肽被切除成的成熟蛋白定位子外膜。现在许多作者利用E.coli分泌蛋白的信号肽序列与目的基因相融合,将外源蛋白分泌出胞质。如用碱性磷酸酶(phoA)的信号肽分泌人α干扰素,使产物的稳定性和产量都有所提高,并且可通过简单的物理技术得到产品。E.coli溶血素通过特殊的分泌机制穿越细胞膜分泌至培养基,现已尝试用该系统构建表达载体。 本文从信号肽的作用、成熟蛋白对分泌的影响、宿主在分泌中的作用、E.coli溶血素的分泌以及外源蛋白在E.coli中的分泌等几方面综述近几年研究E.coli蛋白质分泌的进展。     

信号肽对酵母外源蛋白质分泌效率的影响

根据酵母表达系统在表达外源蛋白方面的独特优势,利用酵母表达系统高效分泌并纯化具有生物学活性的蛋白质,已受到广泛关注。信号肽在蛋白质的分泌中起着重要作用,可引导蛋白分泌至胞外,大大提高蛋白的表达量,在工业化生产外源蛋白的纯化工艺方面具有重要意义。该文将从酵母表达系统中对信号肽的选择、改造、偏爱密码子和增强子的应用等几个方面进行优化的探讨,以提高蛋白质在酵母系统中的分泌效率。     

L-型细菌蛋白表达系统

L-型细胞由于缺乏细胞壁以及具有的其它特殊生物学特性,通过连接合适的信号肽,可以用于许多外源蛋白的可溶性,功能活性形式的分泌表达,表达产物在培养基中,表达的产量依赖于不同的基因序列,载体,宿主和诱导表达条件等因素,此外,L-型细菌还能将具有功能活性的蛋白展示在胞浆膜表面。     

外源蛋白在芽孢杆菌中分泌表达的研究进展

芽孢杆菌是很有潜力的分泌型基因工程宿主菌。本文概述了利用芽孢杆菌分泌表达外源基因时,影响目的蛋白产率的一些主要因素,如蛋白酶水解作用、缺乏适宜的分子伴侣、信号肽的选择不当等,并讨论了相应的解决对策。     

外源蛋白在芽孢杆菌中分泌表达的研究进展

芽孢杆菌是很有潜力的分泌型基因工程宿主菌。本文概述了利用芽孢杆菌分泌表达外源基因时,影响目的蛋白产率的一些主要因素,如蛋白酶水解作用、缺乏适宜的分子伴侣、信号肽的选择不当等,并讨论了相应的解决对策。     

eEF1A1基因克隆、原核分泌表达及融合蛋白纯化

eEF1A1作为蛋白合成中的重要翻译延伸因子,可与多种功能性蛋白如F-actin、BPOZ-2结合,并在细胞凋亡、蛋白降解方面起重要作用.以往原核基因工程蛋白表达系统大多为包涵体表达的变性分子,需要复性.为了获得eEF1A1原核分泌性可溶性蛋白分子,克隆了人eEF1A1蛋白编码序列(约1 300 bp),并成功构建pET22b-A原核分泌表达重组质粒,转化到大肠杆菌BL21(DE3)菌株,0.4 mmol/L终浓度IPTG诱导,经不同温度下包涵体与胞浆蛋白组分分析,快速明确蛋白表达情况,即诱导4 h后,37℃表达于包涵体组分,在30℃分泌表达至胞浆组分.通过His-Trap亲和层析纯化柱进行线性洗脱,Bradford法测定蛋白浓度高达620 mg/mL,SDS-PAGE分析纯度约为95%,蛋白大小符合50 kD,Western blotting显示目的蛋白能被eEF1A1抗体识别;质谱分析证实重组蛋白为人eEF1A1蛋白分子.为进一步研究其与重要功能性蛋白的相互作用及在细胞凋亡和蛋白降解中的作用奠定基础.     

PSP94融合蛋白在大肠杆菌中的表达及其抗前列腺癌活性分析

在从成年人正常前列腺组织中获得人９４个氨基酸的前列腺分泌蛋白（ＰＳＰ９４）ｃＤＮＡ基础上,利用ＰＬ表达系统,实现了人ＰＳＰ９４成熟肽Ｎ 末端带有１９个外源氨基酸的融合蛋白在大肠杆菌中的表达。目的蛋白在细胞中主要以包涵体形式存在,表达量约占菌体总蛋白的３０％,分子量约为１６－５ｋＤ。表达产物在人前列腺癌细胞ＰＣ ３上活性分析表明,该融合蛋白能明显抑制前列腺癌细胞的生长。     

一种表征蛋白质可分泌性的结构融合度特征

选择适宜的信号肽是实现外源蛋白高效分泌表达的一个重要因素。本研究利用生物信息学方法分析信号肽与外源蛋白之间的相容程度,将其定义为结构融合度,并从数学角度分析拼接信号肽与目的蛋白邻近残基之间的相互作用,提出了信号肽拼接区域与目标蛋白之间的数学模型,利用该模型进行结构融合度特征提取,以此来表征外源蛋白质的可分泌性。模拟结果显示结构融合度特征能有效区分枯草芽孢杆菌宿主的可分泌和不可分泌蛋白。研究结果有助于信号肽的选择,对目的蛋白分泌表达的优化具有一定的指导意义。     

Codon Usage in Signal Sequences Affects Protein Expression and Secretion Using Baculovirus/Insect Cell Expression System

By introducing synonymous mutations into the coding sequences of GP64sp and FibHsp signal peptides, the influences of mRNA secondary structure and codon usage of signal sequences on protein expression and secretion were investigated using baculovirus/insect cell expression system. The results showed that mRNA structural stability of the signal sequences was not correlated with the protein production and secretion levels, and FibHsp was more tolerable to codon changes than GP64sp. Codon bias analyses revealed that codons for GP64sp were well de-optimized and contained more non-optimal codons than FibHsp. Synonymous mutations in GP64sp sufficiently increased its average codon usage frequency and resulted in dramatic reduction of the activity and secretion of luciferase. Protein degradation inhibition assay with MG-132 showed that higher codon usage frequency in the signal sequence increased the production as well as the degradation of luciferase protein, indicating that the synonymous codon substitutions in the signal sequence caused misfolding of luciferase instead of slowing down the protein production. Meanwhile, we found that introduction of more non-optimal codons into FibHsp could increase the production and secretion levels of luciferase, which suggested a new strategy to improve the production of secretory proteins in insect cells.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

MMP-9信号肽高效诱导PEX重组蛋白在COS7细胞中分泌表达

为了便于收集和纯化, 重组蛋白常需要引导至真核细胞外。蛋白能否分泌主要取决于其是否含有信号肽, 由于不同信号肽诱导蛋白分泌的效率不同,高效信号肽的筛选已成为生物工程领域提高重组蛋白产量的重要策略之一。为了筛选诱导MMP-2 C末端PEX在COS7细胞中高效分泌表达的信号肽,在PEX的N末端分别融合大鼠生长激素(rGH)、小鼠IgG κ链和人基质金属蛋白酶-9（matrix metalloproteinase 9, MMP-9）的信号肽并比较三种信号肽引导PEX分泌表达的效率。Western免疫印迹和ELISA蛋白定量检测表明MMP-9的信号肽引导PEX蛋白分泌的效率约为其它两种信号肽的两倍。利用Ni-NTA亲和柱对细胞培养基中的PEX进行纯化,蛋白产量约为1mg/L,纯化的PEX重组蛋白具有抑制鸡尿囊膜（chorioallantoic membrane,CAM）血管发生的作用。以上结果提示MMP-9的信号肽有效诱导具有生物活性的PEX重组蛋白在COS7细胞中分泌表达。     

Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function

The function of amino-terminal pro-specific peptides (propeptides), sequences often found on intermediate precursor forms of secreted proteins, is poorly understood. Human preproparathyroid hormone (prepro-PTH), a precursor protein containing such a propeptide, is initially synthesized as a precursor containing a 25-amino acid signal sequence, a 6-amino acid propeptide, and the 84-amino acid mature secreted peptide. Cloned cDNA encoding prepro-PTH and synthetic oligonucleotides were used to generate a mutant missing precisely the pro-specific sequences. The effects of this deletion on signal sequence function and on secretion per se were assessed after expression of the mutant cDNA in intact cells and in a cell-free translation system using synthetic mRNA in the presence of microsomal membranes. The mutant precursor protein was inefficiently translocated and cleaved, and cleavage occurred both at the normal site and within the signal sequence. Thus, for the eukaryotic protein prepro-PTH, sequences immediately downstream and separate from the classically defined signal sequence facilitate accurate and efficient signal function.     

Efficiency of erythropoietin's signal peptide for HIV(MN)-1 gp 120 expression

The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.     

Mammalian homologues of the Drosophila slit protein are ligands of the heparan sulfate proteoglycan glypican-1 in brain.

Using an affinity matrix in which a recombinant glypican-Fc fusion protein expressed in 293 cells was coupled to protein A-Sepharose, we have isolated from rat brain at least two proteins that were detected by SDS-polyacrylamide gel electrophoresis as a single 200-kDa silver-stained band, from which 16 partial peptide sequences were obtained by nano-electrospray tandem mass spectrometry. Mouse expressed sequence tags containing two of these peptides were employed for oligonucleotide design and synthesis of probes by polymerase chain reaction and enabled us to isolate from a rat brain cDNA library a 4.1-kilobase clone that encoded two of our peptide sequences and represented the N-terminal portion of a protein containing a signal peptide and three leucine-rich repeats. Comparisons with recently published sequences also showed that our peptides were derived from proteins that are members of the Slit/MEGF protein family, which share a number of structural features such as N-terminal leucine-rich repeats and C-terminal epidermal growth factor-like motifs, and in Drosophila Slit is necessary for the development of midline glia and commissural axon pathways. All of the five known rat and human Slit proteins contain 1523-1534 amino acids, and our peptide sequences correspond best to those present in human Slit-1 and Slit-2. Binding of these ligands to the glypican-Fc fusion protein requires the presence of the heparan sulfate chains, but the interaction appears to be relatively specific for glypican-1 insofar as no other identified heparin-binding proteins were isolated using our affinity matrix. Northern analysis demonstrated the presence of two mRNA species of 8. 6 and 7.5 kilobase pairs using probes based on both N- and C-terminal sequences, and in situ hybridization histochemistry showed that these glypican-1 ligands are synthesized by neurons, such as hippocampal pyramidal cells and cerebellar granule cells, where we have previously also demonstrated glypican-1 mRNA and immunoreactivity. Our results therefore indicate that Slit family proteins are functional ligands of glypican-1 in nervous tissue and suggest that their interactions may be critical for certain stages of central nervous system histogenesis.     

Signal-sequence Trap in Mammalian and Yeast Cells: A Comparison

Membrane associated and secreted proteins are translated as precursors containing a signal peptide that allows protein-insertion into the membrane of the endoplasmic reticulum and is co-translationally removed in the lumen. The ability of the signal peptide to direct a polypeptide into the secretory pathway is exploited in methods developed to select cDNAs encoding such proteins. Different strategies are known in which cDNA libraries can be screened for signal peptides by the ability of the latter to rescue the translocation of signal sequence-less proteins. In one method, a cDNA library is tested for interleukin 2 receptor α chain translocation to the membrane in COS cells, in another one for invertase secretion from yeast. In this work, we compared the two systems by testing six mouse signal peptides in COS and yeast cells. All of them were functional in the mammalian system, whereas only three of them in yeast. Two other sequences needed the 5′ cDNA sequence flanking the ATG codon to be removed in order to enable protein translocation. Although the structure of signal sequences and the functioning of the secretory machinery are well conserved from prokaryotes to eukaryotes, it seems evident that not all signal peptides can be interchanged between different proteins and organisms. In particular, signal peptides that are functional in the mammalian system do not necessarily lead to protein translocation in yeast. Received: 9 March 2001