内含肽介导的生物学效应及其应用

蛋白质翻译产物在成熟过程中剪切释放出来的一段氨基酸序列称为“intein”---即内含肽。它与前体蛋白以框内融合的形式共同翻译,并内嵌于前体蛋白序列中。内含肽的解离以及内含肽两侧氨基酸序列的连接是在内含肽自身催化作用下完成的。本文将从内含肽的发现、结构特征和作用机理等方面对这种具有特殊意义的蛋白质成熟机制进行较为全面的论述,同时介绍了近年来发展起来的以内含肽介导的蛋白质剪接为基础的蛋白质纯化和改造技术。     

内含肽介导的蛋白连接技术及其应用

内含肽介导的蛋白质剪接是一种自发的翻译后事件,内含肽可介导其自身从前体蛋白上切除,同时将其两侧的外显肽连接起来.在过去10多年中,基于蛋白质剪接原理发展出的蛋白连接技术被广泛的用于蛋白质工程的研究中.这些技术打破了化学合成方法中对目标物大小的限制,有助于化学和生物学的研究.针对近年由蛋白质连接演化出来的新技术及其应用做简要的阐述.     

NMR resonance assignment of DnaE intein from Nostoc punctiforme

DnaE intein from Nostoc punctiforme (Npu) is one of naturally occurring split inteins, which has robust protein splicing activity. Highly efficient trans-splicing activity of NpuDnaE intein could widen various biotechnological applications. However, structural basis of the efficient protein splicing activity is poorly understood. As a first step toward better understanding of protein trans-splicing mechanism, we present the backbone and side-chain resonance assignments of a single chain variant NpuDnaE intein as determined by triple resonance experiments with [¹³C,¹⁵N]-labeled protein.     

Purification of green fluorescent protein using a two-intein system

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.     

Isolation and characterization of bacteriophage PM2 from Aeromonas hydrophila

PM2 is an Aeromonas-specific bacteriophage isolated on A. hydrophila strain AH-3. The bacteriophage receptor for this phage was found to be the lipopolysaccharide (LPS), specifically a low-molecular weight LPS fraction (LPS-core oligosaccharides). Mutants resistant to this phage were isolated and found to be devoid of LPS O-antigen and altered in the LPS-core. No other outer-membrane (OM) molecules appeared to be involved in phage binding.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

Characterization of an Actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila

The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler''s diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K_m(NAD⁺) = 6 μm, K_m(actin) = 24 μm, and k_cat = 22 s⁻¹. VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 Å crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC₅₀ = 20 μm). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce.

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

1H, 13C, and 15N NMR assignments of an engineered intein based on Mycobacterium tuberculosis RecA

The backbone and side chain resonance assignments of an engineered intein based on Mycobacterium tuberculosis RecA have been determined based on triple-resonance experiments with the uniformly [¹³C,¹⁵N]-labeled protein.     

Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila

Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6xHis-rGalE was determined to be 38271.477 (m/z). The 6xHis-rGalE with a K(m) of 0.5 mM for UDP-galactose exhibited optimum activity at 37 degrees C and pH 8-9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.     

Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin

The intein that interrupts the DNA polymerase II DP2 subunit in Pyrococcus abyssi can be overexpressed in Escherichia coli and purified as an unspliced precursor. On in vitro incubation at 37 degrees Celsius or higher, the intein mediates efficient protein splicing. Mutations can be introduced into an intein fusion protein that prevent the second and third steps of protein splicing. As a result, the intein fusion protein can facilitate temperature-dependent formation of a thioester linkage between the N-extein and intein. This thioester is susceptible to in vitro hydrolysis or thiolysis at temperatures of 40 degrees Celsius or higher, and we have exploited this activity to generate a temperature-dependent protein purification scheme. Protein purification using this intein does not require the addition of exogenous thiols and is compatible with the use of immobilized metal affinity chromatography. The identity of the C-terminal residue of the N-extein has less influence on the cleavage reaction than in current purification systems in terms of premature in vivo cleavage and is complementary to current systems in terms of efficient in vitro cleavage.     

Kinetic, spectroscopic, and X-ray crystallographic characterization of the functional E151H aminopeptidase from Aeromonas proteolytica

Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the D-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in DppA. Substitution of E151 with histidine resulted in an active AAP enzyme exhibiting a kcat value of 2.0 min(-1), which is over 2000 times slower than r AAP (4380 min(-1)). ITC experiments revealed that ZnII binds 330 and 3 times more weakly to E151H-AAP compared to r-AAP. UV-vis and EPR spectra of CoII-loaded E151H-AAP indicated that the first metal ion resides in a hexacoordinate/pentacoordinate equilibrium environment, whereas the second metal ion is six-coordinate. pH dependence of the kinetic parameters kcat and K(m) for the hydrolysis of L-leucine p-nitroanilide (L-pNA) revealed a change in an ionization constant in the enzyme-substrate complex from 5.3 in r-AAP to 6.4 in E151H-AAP, consistent with E151 in AAP being the active-site general acid/base. Proton inventory studies at pH 8.50 indicate the transfer of one proton in the rate-limiting step of the reaction. Moreover, the X-ray crystal structure of [ZnZn(E151H-AAP)] has been solved to 1.9 A resolution, and alteration of E151 to histidine does not introduce any major conformational changes to the overall protein structure or the dinuclear ZnII active site. Therefore, a histidine residue can function as the general acid/base in hydrolysis reactions of peptides and, through analogy of the role of E151 in AAP, H115 in DppA likely shuttles a proton to the leaving group of the substrate.     

Functional analysis of the split Synechocystis DnaE intein in plant tissues by biolistic particle bombardment

The DnaE intein of Synechocystis sp. PCC6803 (Ssp DnaE intein) is the first split intein identified in nature. Its N-terminal fragment (Int-n) is attached to the end of the N-terminal half of the DnaE protein (DnaE-n) to form the precursor DnaE-n/Int-n, while the C-terminal fragment (Int-c) precedes the C-terminal half of the DnaE protein (DnaE-c) to form the precursor Int-c/DnaE-c. Int-n and Int-c fragments in the separate precursors catalyze, in concert, a protein trans-splicing process to splice the flanking DnaE-n and DnaE-c into a functional catalytic subunit of DNA polymerase III. They then release themselves from the precursors. Previously, the Ssp DnaE intein has been used to reconstitute a protein trans-splicing mechanism in stably transformed Arabidopsis thaliana, resulting in successful reassembly of an intact and functional GUS from two halves of a split GUS protein. In this report, transient expression using a biolistic particle bombardment approach is described for functional analysis of Ssp DnaE intein. Analyses confirmed that the Ssp DnaE intein could catalyze protein trans-splicing not only in model plants but also in monocot and dicot crops. It also demonstrated that when up to 45 amino acid residues were removed from the C-terminus of the Int-n fragment, the Int-n fragment was still able to function in the protein trans-splicing process.     

Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila.

A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product.     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.     

Kinetic and structural characterization of reversibly inactivated beta-lactamase

The reversible inhibition of beta-lactamase I from Bacillus cereus by cloxacillin, methicillin, and nafcillin has been systematically investigated. For these substrates the enzymatic reaction involves partitioning of the substrate between turnover and inhibition. Typically, concentrations of several hundred millimolar are necessary for complete inactivation. The completely inactivated enzyme could be formed by incubation at temperatures above 20 degrees C, where inhibition competes more effectively with turnover, and then stabilized by dropping the temperature to 0 degrees C or lower. The inactivated enzyme was rapidly separated from unreacted substrate and product at low temperature by centrifugal gel filtration or ion exchange and examined by far-UV circular dichroism for evidence of a conformational change. At pH 7 the inactivated enzyme had a secondary structure essentially identical with that of the native enzyme. The fluorescence emission spectrum of the inactivated enzyme (at pH 7) was also identical with that of the native enzyme. However, the inactivated enzyme was found to be considerably more sensitive to thermal denaturation, to acid-induced conformational isomerization, and to trypsinolysis than the native enzyme. We conclude from the circular dichroism results that the structure of the reversibly inactivated enzyme cannot be significantly different from that of the native enzyme. Therefore, previous findings that have been interpreted as indicating a major conformational change must be reevaluated. From examination of the low-resolution crystallographic structure of the enzyme we propose that the most likely cause of the inactivation is an alternate conformational state of the acyl-enzyme intermediate involving movement of one or more of the alpha-helices forming part of the active site. Such a structural effect could leave the secondary structure unchanged but have significant effects on the tertiary structure, catalysis, mobility, and susceptibility to trypsin and denaturation. We propose that the underlying physical reason why certain beta-lactam substrates bring about this "substrate-induced deactivation", or suicide inactivation, of the enzyme is due to the presence of the alternative acyl-enzyme conformation of similar free energy to the productive one, in which one (or more) essential catalytic group is no longer optimally oriented for catalyzing deacylation. Thus for substrates with a slow rate of deacylation (less than or equal to 100 s-1) the conformational transition can compete effectively on the time scale of the turnover reaction.     

Cloning and characterization of an extracellular temperature-labile serine protease gene from Aeromonas hydrophila

Aeromonas virulence is thought to depend on multigenic functions. The gene for an extracellular protease from Aeromonas hydrophila SO2/2 was cloned in Escherichia coli C600-1 by using pIJ860, bifunctional plasmid, as a vector. The gene encodes for a temperature-labile serine protease (P2) with a molecular mass of approx. 68 kDa which is highly inhibited by PMSF. The gene was expressed in Streptomyces lividans 1326 by transforming protoplasts with the original clone pPA2. We were also able to transfer and express the prt P2 gene in Pseudomonas putida by mating experiments. The protein P2 was secreted into the periplasms of both P. putida and E. coli C600-1 being identical in properties to one of the proteases secreted into the culture supernatant by A. hydrophila SO2/2.     

嗜水气单胞菌J-1株弹性蛋白酶的表达、纯化及特性分析

摘要：【目的】表达、纯化嗜水气单胞菌J-1株弹性蛋白酶,并对弹性蛋白酶的性质进行分析。【方法】以pET-32a为表达载体将弹性蛋白酶基因ahyB转化至大肠杆菌BL21菌株中进行诱导表达,表达重组酶用His TaqNi2+亲和层析柱纯化并用6 mol/L盐酸胍进行复性;利用硫酸铵分级沉淀、阴离子交换层析和分子筛层析对嗜水气单胞菌培养上清液中的弹性蛋白酶进行纯化。将【结果】从嗜水气单胞菌培养上清液中获得的弹性蛋白酶原酶的最适pH 为8.5,而表达重组酶为 10.0;对热的稳定性,原酶高于表达酶。两种形式酶的性     

嗜水气单胞菌若干毒力因子同时缺失的突变株的构建及特性分析

以携带质粒pAM12 0 (Tcr Tn916 )的大肠杆菌CG12 0株为供体菌 ,采用滤膜接合法与受体菌嗜水气单胞菌J_1株 (cfzr)进行接合转移 ,在含Tc和cfz选择平板上进行筛选。共获接合转移菌落 380 0个 ,其接合频率为 3× 10 - 5(按供体细胞计算 )。任取 38个接合子 ,提取基因组DNA ,以嗜水气单胞菌特异性 16SrDNA引物进行PCR扩增 ,所有接合子均阳性。为证明Tn916确实插入基因组 ,以四环素基因 (tet)引物进行PCR扩增 ,结果所有抗性接合子均扩增出一条特异条带。与亲本J_1株相比 ,所有接合子的主要毒力因子如蛋白酶、溶血素、DNA酶和淀粉酶等均不表达 ,对小鼠失去致病力 ,其LD50 大于 10 9CFU。接合子连传 10次后 ,四环素抗性消失 ,但毒力未恢复 ,说明通过转座子Tn916的插入可获得稳定的无毒嗜水气单胞菌突变株。Tn916引起嗜水气单胞菌毒力性状改变的机制有待研究 ,推测可能与该菌染色体上存在Tn916的热点或毒力岛有关。