Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli

Human beta-defensin-2 (hBD2) is a cysteine-rich cationic antimicrobial peptide with low molecular weight that exhibits a broad range of antimicrobial activity. To improve the expression level of hBD2 in Escherichia coli, tandem repeats of mature hBD2 gene were constructed and expressed as fusion proteins (TrxA-nmhBD2, n=1, 2, 4, 8) by constructing the vectors of pET32-nsmhBD2 (n=1, 2, 4, 8). The results showed that the tandem repeats of mhBD2 gene were highly expressed in our constructed system. Comparing the expression levels of soluble mhBD2, BL21(DE3)/pET32-2smhBD2 was selected as an ideal recombinant strain for mature hBD2 production. Under the optimized conditions of cultivation and isopropylthiogalactoside (IPTG) induction, the maximum expression level of soluble mature hBD2 (0.76 g/l) with the highest percentage of fusion protein in soluble proteins (62.2%) was obtained in the present work, which was the highest yield of hBD2 reported so far.     

大腹园蛛次壶腹腺丝的表达

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道,研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1,融合his-tag标签,构建酵母表达载体,在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体,在大肠杆菌BL21(DE3)中进行表达。SDS-PAGE和Western blotting检测结果表明,P1在两种表达系统中均可实现表达。研究结果显示:P1在GS115中的表达经优化后产量、产率有较大提高,且远高于BL21(DE3)中的表达,相应的纯化效率GS115也远高于对照BL21(DE3)的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达,为表达全长天然MiSp编码序列提供前期实验基础,也为大规模蛛丝蛋白的重组表达建立了平台。     

Alternative topogenic signals in peroxisomal citrate synthase of Saccharomyces cerevisiae.

The tripeptide serine-lysine-leucine (SKL) occurs at the carboxyl terminus of many peroxisomal proteins and serves as a peroxisomal targeting signal. Saccharomyces cerevisiae has two isozymes of citrate synthase. The peroxisomal form, encoded by CIT2, terminates in SKL, while the mitochondrial form, encoded by CIT1, begins with an amino-terminal mitochondrial signal sequence and ends in SKN. We analyzed the importance of SKL as a topogenic signal for citrate synthase, using oleate to induce peroxisomes and density gradients to fractionate organelles. Our experiments revealed that SKL was necessary for directing citrate synthase to peroxisomes. C-terminal SKL was also sufficient to target a leaderless version of mitochondrial citrate synthase to peroxisomes. Deleting this tripeptide from the CIT2 protein caused peroxisomal citrate synthase to be missorted to mitochondria. These experiments suggest that the CIT2 protein contains a cryptic mitochondrial targeting signal.     

Development and application of an in vivo plant peroxisome import system.

The purposes of this study are to develop an in vivo cell system that is suitable for the immunofluorescent detection of transiently expressed proteins targeted to plant peroxisomes and to determine whether a C-terminal serine-lysine-leucine (SKL) tripeptide, a consensus-targeting signal for mammalian peroxisomes, also targets proteins to plant peroxisomes. Protoplasts from mesophyll cells and from suspension-cultured cells initially were examined for their potential as an in vivo import system. Several were found suitable, but based on a combination of criteria, suspension-cultured tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells (TBY-2) were chosen. The tobacco cell extracts had catalase activity, and two polypeptides of approximately 55 and 57 kD specifically were detected on immunoblots with anti-cottonseed catalase immunoglobulins G as the probe. Indirect immunofluorescence microscopy with these immunoglobulins G revealed a punctate labeling pattern indicative of endogenous catalase localization within putative TBY-2 peroxisomes. The cells did not have to be completely converted to protoplasts for optimal microscopy; treatment with 0.1% (w/v) pectolyase for 2 h was sufficient. Microprojectile bombardment proved superior for transient transformation of the TBY-2 cells with plasmids encoding beta-glucuronidase, or chloramphenicol acetyltransferase (CAT), or CAT with an added C-terminal tripeptide (CAT-SKL). C-terminal SKL is a consensus, type 1, peroxisome targeting signal. Double indirect immunofluorescent labeling showed that CAT-SKL co-localized with endogenous catalase. Non-punctate, diffuse localization of CAT without SKL provided direct evidence that the C-terminal SKL tripeptide was necessary and sufficient for targeting of CAT to plant peroxisomes. These data demonstrate the effectiveness of this peroxisome targeting signal for plant cells.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes

Shock wave treatment (SWT) was shown to induce regeneration of ischaemic myocardium via Toll‐like receptor 3 (TLR3). The antimicrobial peptide LL37 gets released by mechanical stress and is known to form complexes with nucleic acids thus activating Toll‐like receptors. We suggested that SWT in the acute setting prevents from the development of heart failure via RNA/protein release. Myocardial infarction in mice was induced followed by subsequent SWT. Heart function was assessed 4 weeks later via transthoracic echocardiography and pressure–volume measurements. Human umbilical vein endothelial cells (HUVECs) were treated with SWT in the presence of RNase and proteinase and analysed for proliferation, tube formation and LL37 expression. RNA release and uptake after SWT was evaluated. We found significantly improved cardiac function after SWT. SWT resulted in significantly higher numbers of capillaries and arterioles and less left ventricular fibrosis. Supernatants of treated cells activated TLR3 reporter cells. Analysis of the supernatant revealed increased RNA levels. The effect could not be abolished by pre‐treatment of the supernatant with RNase, but only by a sequential digestion with proteinase and RNase hinting strongly towards the involvement of RNA/protein complexes. Indeed, LL37 expression as well as cellular RNA uptake were significantly increased after SWT. We show for the first time that SWT prevents from left ventricular remodelling and cardiac dysfunction via RNA/protein complex release and subsequent induction of angiogenesis. It might therefore develop a potent regenerative treatment alternative for ischaemic heart disease.     

玉米大斑病菌STK1与EGFP融合基因载体的构建及其在毕赤酵母中的表达

STK1基因是玉米大斑病菌调控分生孢子发育、渗透胁迫调节和致病性的重要MAPK基因。本文首先构建了含有增强型绿色荧光蛋白基因(EGFP)的毕赤酵母GSS115(Pichia pastoris GS115)表达载体p PIC3.5K-EGFP,再以玉米大班病菌模式菌株01-23的菌丝c DNA为模板,PCR扩增STK1基因,克隆到p PIC3.5K-EGFP,构建了STK1-EGFP融合基因的GS115表达载体p PIC3.5K-STK1-EGFP。利用电击转化法将该融合基因表达载体转化到GS115感受态细胞内,利用MD培养基筛选、PCR鉴定,获得了STK1-EGFP融合基因的毕赤酵母转化子。通过RT-PCR和荧光观察,发现STK1基因和EGFP基因均可以高效稳定地表达。另外,在试验中我们还发现,在STK1基因起始密码子前加入Kozak序列可以使STK1-EGFP融合基因的表达强度增强4.8倍。以上研究结果为STK1基因表达蛋白的亚细胞功能定位和抗体制备奠定了基础。     

Transport of chimeric proteins that contain a carboxy-terminal targeting signal into plant microbodies

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.     

两株绿脓杆菌对石油污染土壤的修复作用

本文旨在研究环境条件下微生物对石油污染土壤的修复情况。从矿井周边土样定向筛选出两株绿脓杆菌,摇瓶降解实验发现,两菌混合培养10 d原油降解率达到95.67%,比单菌培养提高至少32%,即两菌对原油降解具有协同作用。根据降解实验结果制备了混合修复菌剂,并且人工构建石油污染场地,展开中试场地修复试验,模拟不同的操作条件下土壤中原油的降解情况。经60 d修复发现,添加了菌剂的场地,石油烃含量下降趋势明显,每克土壤中石油烃含量从初始的0.8%降至0.1%–0.3%,其中额外添加有机肥作为补充碳氮源的场地,总石油烃降解率最高,达到85.28%。而未添加菌剂的对照组石油烃含量仅减少25.85%。     

High-level expression of a biologically active staphylokinase in Pichia pastoris

Staphylokinase (SAK) as the third generation thrombolytic molecule is a promising agent for the treatment of thrombosis. SAK variant of SAKфC was expressed in Pichia pastoris strains KM71H and GS115. The codon adaptation index of SAK was improved from 0.75 to 0.89. The expression of recombinant SAK (rSAK) reached to its maximum (310?mg/L of the culture medium) after 48-hr stimulation with 3% methanol and remained steady until day 5. The maximum activity of the enzyme was at pH 8.6 and 37°C. It was highly active at temperatures 20–37°C and pH ranges of 6.8–9 (relative residual activity more than 80%). It was determined that rSAK was 73.8% of the total proteins secreted by P. pastoris KM71H into the culture media. The specific activities of rSAK were measured as 9,002 and 21,042?U/mg for the nonpurified and purified proteins, respectively. The quantity of the purified protein (>99% purity) was 720?µg/mL with a purification factor of 2.34. Western blot analysis showed two bands of nearly 22 and 18.6?kDa. It was concluded that P. pastoris is a proper host for expression of biologically active and endotoxin-free rSAK due to its high expression and low protein impurity in culture supernatant.     

快速筛选高效表达重组蛋白毕赤酵母菌株新方法的建立及评价

毕赤酵母是当前应用最为广泛的重组蛋白表达系统之一,文中建立了一种快速筛选高效表达重组蛋白的毕赤酵母菌株的新方法。首先,对内质网转膜蛋白Sec63融合表达增强型绿色荧光蛋白EGFP的改造菌株GS115-E表达重组蛋白的能力进行检测;之后将携带不同拷贝数的植酸酶phy基因或木聚糖酶xyn基因的质粒转化进入GS115-E中,得到具有不同植酸酶或木聚糖酶表达水平的重组菌株,分别检测不同菌株的EGFP与重组蛋白的表达水平;最后,利用分选型流式细胞仪,根据绿色荧光值的高低对包含不同植酸酶表达水平的重组菌株的菌群进行分选。结果显示重组菌株中EGFP的荧光值与重组蛋白的活性表达水平之间具有良好的线性相关性(0.8|R|1),且利用流式细胞仪可高效地从混合菌群中筛选得到高产菌株,所分选得到的高荧光菌株在摇瓶发酵120 h时植酸酶表达水平是低荧光菌株的4.09倍。本方法通过检测菌株的EGFP荧光值代替检测重组蛋白的表达水平和活性,从而实现高表达菌株的筛选,大大提高了其应用的便捷性及通用性。与流式细胞仪、液滴微流控等高通量筛选仪器或技术结合将进一步提高筛选的速度与通量,为筛选获得高效表达重组蛋白的毕赤酵母菌株提供了简便、快速的新途径。     

Targeting expression of the catalytic domain of the kinase insert domain receptor (KDR) in the peroxisomes of Pichia pastoris

The kinase insert domain receptor (KDR), also known as vascular endothelial growth factor receptor-2 (VEGFR2), is an important therapeutic target for the treatment of cancer because of its crucial role in angiogenesis, which is fundamental to the malignancy of tumors. Here, we expressed the catalytic domain of KDR in Pichia pastoris under the control of the AOX1 promoter. In order to facilitate its purification and detection, His-tag and green fluorescent protein (GFP) were fused to the N-terminus of KDR. At the same time, a peroxisomal targeting signal 1 (SKL) was fused to the C-terminus to avoid the potential negative effect on the host cell. The highly expressing clone K1 was selected by GFP fluorescence intensity analysis using flow cytometry (FCM). Furthermore, the GFP-KDR-SKL fusion protein was proved to be correctly targeted to the peroxisomes of P. pastoris by colocation with blue fluorescent protein-SKL. The expression of GFP-KDR-SKL led to extensive phosphorylation of endogenous proteins and significantly inhibited cell growth. However, the expression was not lethal to the cells. Both in vitro biological activity assay and inhibition rate assay demonstrated that the purified GFP-KDR-SKL fusion protein exhibited high kinase catalytic activity and could be used as a target for anticancer drug screening.     

C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

Summary The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 m diameter), a characteristic of so-called unspecialized peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to -ATPase and stearoyl-ACP 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.Abbreviations CAT chloramphenicol acetyltransferase - CHO cells Chinese hamster ovary cells - DAB 3,3-diaminobenzidine - GUS -glucuronidase - ICL isocitrate lyase - KSRM lysine-serine-arginine-methionine - MS malate synthase - PBS phosphate-buffered saline - PTS peroxisome targeting signal - SKL serine-lysine-leucine - tobacco BY-2 Bright Yellow-2 Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD_cenA) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.     

PTD-NPY融合基因的克隆及其在毕赤酵母中的分泌表达

应用重叠延伸PCR方法扩增HIV-1 TAT蛋白转导结构域(PTD)与鼠源神经肽Y(NPY)的融合基因,克隆目的片段并插入酵母表达载体pPICZαA,构建成重组表达质粒pPICZα-PTD-NPY.PCR和酶切鉴定及测序正确后,经限制性内切酶Sac Ⅰ线性化重组表达质粒并通过电转化整合到巴斯德毕赤酵母菌GS115的染色体基因组中.阳性重组酵母菌用含1%甲醇的培养基诱导其分泌表达.经过120 h的诱导,取上清浓缩除盐后进行SDS-PAGE电泳,表明该系统成功表达了PTD-NPY融合蛋白,Western blotting实验证实表达产物具有特异性.获得真核表达的PTD-NPY融合蛋白,为下一步的应用研究提供了物质基础.     

Control of apple blue mold by <Emphasis Type="Italic">Pichia pastoris</Emphasis> recombinant strains expressing cecropin A

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.     

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320- 322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47- dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.     

Cytosol-dependent peroxisomal protein import in a permeabilized cell system

Using streptolysin-O (SLO) we have developed a permeabilized cell system retaining the competence to import proteins into peroxisomes. We used luciferase and albumin conjugated with a peptide ending in the peroxisomal targeting sequence, SKL, to monitor the import of proteins into peroxisomes. After incubation with SLO-permeabilized cells, these exogenous proteins accumulated within catalase-containing vesicles. The import was strictly signal dependent and could be blocked by a 10-fold excess of peptide containing the SKL-targeting signal, while a control peptide did not affect the import. Peroxisomal accumulation of proteins was time and temperature dependent and required ATP hydrolysis. Dissipation of the membrane potential did not alter the import efficiency. GTP-hydrolyzing proteins were not required for peroxisomal protein targeting. Depletion of endogenous cytosol from permeabilized cells abolished the competence to import proteins into peroxisomes but import was reconstituted by the addition of external cytosol. We present evidence that cytosol contains factors with SKL-specific binding sites. The activity of cytosol is insensitive to N- ethylmaleimide (NEM) treatment, while the cells contain NEM-sensitive membrane-bound or associated proteins which are involved in the import machinery. The cytosol dependence and NEM-sensitivity of peroxisomal protein import should facilitate the purification of proteins involved in the import of proteins into peroxisomes.     

Biologically active and C-amidated hinnavinII-38-Asn produced from a Trx fusion construct in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15–20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.