Functional expression in pichia pastoris of an acidic pectin methylesterase from jelly fig (Ficus awkeotsang)

A cDNA fragment encoding an acidic pectin methylesterase (PME) of jelly fig achene was successfully expressed in Pichia pastoris under the control of the glyceraldehydes-3-phosphate dehydrogenase promoter. The recombinant PME was produced as a secretory protein by N-terminal fusion of a cleavable prepropeptide for signal trafficking, and thus easily harvested from the culture medium. Compared with native N-glycosylated PME (38 kDa) purified from jelly fig achenes, this recombinant PME (45 kDa) appeared to be hyperglycosylated. Activity staining indicated that the recombinant PME was functionally active. Yet the hyperglycosylated recombinant PME possessed thermostability and enzymatic capability over a broad pH range equivalent to those of the native PME. The success of functional production of this acidic jelly fig PME in P. pastoris has significantly broadened its applications in industry.     

Purification, cloning, and identification of two thaumatin-like protein isoforms in jelly fig (Ficus awkeotsang) Achenes

Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of beta-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments.     

Purification and glycosylation analysis of an acidic pectin methylesterase in jelly fig (Ficus awkeotsang) achenes

An acidic pectin methylesterase (PME) is responsible for the gelation of water extract from jelly fig (Ficus awkeotasang) achenes. A new, fast and efficient, method has been developed to purify this acidic PME. The method includes preparing jelly curd by traditional hand washing, extracting proteins from the curd, and separating PME by anion-exchanger. The purified PME exists as a monomer of 38 kDa determined by gel filtration, and exerts enzymatic activity over a broad pH range, particularly in acidic environments where most known PME enzymes from various species are inactivated. Chemical staining and enzymatic cleavage suggest that the jelly fig PME is an N-linked glycoprotein. Fluorophore-assisted carbohydrate electrophoresis reveals that the polysaccharide of this glycoprotein putatively consists of 22 hexoses including 16 mannose, 4 N-acetylglucosamine, and 2 galactose residues.     

Characterization of pectinesterase inhibitor in jelly fig (Ficus awkeotsang Makino) achenes

Pectinesterase inhibitor (PEI) extract prepared from intact jelly fig (Ficus awkeotsang Makino) achenes was separated by membrane (MWCO 3 and 10 kDa) and fractionated by a Sepharose G-50 gel permeation chromatography. Results from Sepharose G-50 gel permeation chromatography and concanavalin A Sepharose chromatography revealed PEI as polypeptides with molecular weights ranging from 3.5 to 4.5 kDa. Incubation of a PE (1 unit/mL)-PEI (2 mg/mL) mixture for 1 min decreased the PE activity by approximately 50%. On the basis of the results of Lineweaver-Burk double-reciprocal plots, Michaelis constant (K(m)) and V(max) values for jelly fig achenes PE (pH 6.0, 30 degrees C) were 0.78 mM -OCH3 and 1.09 microequiv of -COOH/min, respectively. In addition, PEI competitively inhibited both citrus and jelly fig achenes PEs.     

Gene cloning and characterization of a novel recombinant antifungal chitinase from papaya (Carica papaya)

A chitinase cDNA clone (CpCHI, 1002 bp) was isolated from papaya fruit, which encoded a 275 amino acid protein containing a 28 amino acid signal peptide in the N-terminal end. The predicted molecular mass of the mature protein was 26.2 kDa, and its pI value was 6.32. On the basis of its amino acid sequence homology with other plant chitinases, it was classified as a class IV chitinase. An active recombinant CpCHI enzyme was overexpressed in Escherichia coli. The purified recombinant papaya chitinase showed an optimal reaction temperature at 30 degrees C and a broad optimal pH ranging from 5.0 to 9.0. The recombinant enzyme was quite stable, retaining >64% activity for 3 weeks at 30 degrees C. The spore germination of Alternaria brassicicola could be completely inhibited by a 76 nM level of recombinant CpCHI. Recombinant CpCHI also showed antibacterial activity in which 50% of E. coli was inhibited by a 2.5 microM concentration of the enzyme.     

Expression of recombinant mature human tyrosinase from Escherichia coli and exhibition of its activity without phosphorylation or glycosylation

A cDNA encoding mature human tyrosinase was cloned into pET-23a(+) and transformed into E. coli BL21(DE3). Three major recombinant proteins, mature human tyrosinase (RHT??????), N-terminal truncated human tyrosinase (RHT???????), and β-lactamase, were overexpressed as inclusion bodies in E. coli after 12 h of induction with 1.0 mM isopropyl-β-D-thiogalactopyranoside at 37 °C. After sonication and centrifugation, the inclusion body was harvested, solubilized, dialyzed, and refolded into the active form with monophenolase and diphenolase activities. It was purified to homogeneity by DEAE-Sepharose FF and Sephadex G-75. The molecular mass and N-terminal sequence were 57.0 kDa and GHFPRAC, respectively, and corresponded to those of mature human tyrosinase. The RHT was active in a broad range of temperature and pH, and with optimum activity at 70 °C and pH 8.5.     

Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase

L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.     

啤酒糖酵母菌6-磷酸海藻糖合成酶编码基因tps1的cDNA克隆

本工作从抗逆性极强的啤酒糖酵母菌株 Ａ Ｓ２１４１６ 中分离纯化总 Ｒ Ｎ Ａ 和 ｍ Ｒ Ｎ Ａ ,以 Ａ Ｍ Ｖ 逆转录酶合成了单链ｃ Ｄ Ｎ Ａ 。采用保守引物扩增并克隆了该酵母菌的６ － 磷酸海藻糖合成酶编码基因ｔｐｓ１ ,建立了该基因 Ｄ Ｎ Ａ 片段的物理图谱,发现其酶切位点与已见报道的６ － 磷酸海藻糖合成酶编码基因相同。     

牛杀菌/通透性增加蛋白氮端片段的原核表达

目的 克隆牛杀菌/通透性增强蛋白(BPI)N端cDNA,构建原核表达载体,在大肠杆菌中表达BPI蛋白,并纯化重组蛋白。方法 参照Genbank报道的序列,应用RT-PCR技术,从牛嗜中性粒细胞mRNA中扩增出杀菌/通透性增加蛋白基因,然后将该基因插入原核表达载体pGEX-4T-1中,重组质粒转化大肠杆菌BL21,进行诱导表达。结果 获得BPI N端长度为714 bp的基因片断,序列分析证实该片断中有1个点突变。大肠杆菌中的表达产物为相对分子质量约为52×103的GST-BPI融合蛋白。结论 成功的表达和纯化了BPI重组蛋白。     

Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis

In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.     

Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification

Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).     

苹果基因邻近未知序列的PCR扩增与测序方法

本文提出的方法是首先利用已知基因序列设计三个巢式PCR引物p14、p15和p16,然后设计3’端为常见酶切位点、5’端为随机引物序列p17的9个酶切位点引物,用p14外引物与9个酶切位点引物进行第一轮PCR扩增,其中前5个反应的退火温度较低可以将酶切位点引物的5’端序列引入到PCR产物中,其余反应则以此PCR产物为模板采用较高的正常退火温度进行,退火温度较高是为了使整个酶切位点引物能稳定地结合到最初反应形成的产物上。为提高PCR产物的特异性和产量,用p15和p16引物分别与p17引物配对进行第二轮和第三轮PCR扩增,电泳检查发现PCR产物条带后进行纯化和测序。为保证序列的正确性,利用测得的DNA序列再设计新引物f3x与p14(或p15)配对,直接用提取的DNA为模板进行PCR扩增和测序。利用此方法成功地获得了苹果FPPS基因邻近的启动子序列528bp和5’UTR序列142bp,并已在GenBank注册（登录号FJ263960）。利用Blastn发现这是GenBank中首次发表苹果FPPS基因启动子序列。这说明用该方法获取基因邻近的未知序列是可行的。     

鸡MHC I α-生物素化序列融合基因的构建、表达及纯化

实验克隆了莱航鸡(Gallus gallus )MHC I α (BF2)(GenBank登陆号：AY989897)全基因，分析了其信号肽序列，构建了缺失信号肽且拼接了BirA底物肽序列(BSP)的融合蛋白重组表达载体pET-BF2-BSP，在大肠杆菌(Escherichia coli )中得到表达，并优化了诱导剂浓度、起始诱导菌体浓度及诱导时间等表达条件。SDS-PAGE分析结果表明所得融合目的蛋白约为40 kD，Western-blot结果显示该重组蛋白成功融合了6×His标签；pET-BF2-BSP最优化表达条件分别为：菌体起始诱导浓度OD600nm 0.8~1.2，IPTG诱导浓度1.1 mmol/L，诱导时间2~4 h；建立了该重组蛋白变性状态下通过镍柱亲和层析纯化包涵体的方法。实验获得了高纯度的在体外构建鸡MHC-肽四聚体所需的重组蛋白BF2-BSP。     

甘薯肌醇-1-磷酸合成酶基因的克隆及序列分析

甘薯(Ipomoea batatas (L.) Lam.)新品系农大603是从感茎线虫(Ditylenchus destructor)病品种徐薯18的辐照后代中获得的一个抗茎线虫病的突变体。以农大603和徐薯18块根的mRNA为模板，根据植物抗线虫病基因NBS保守氨基酸序列设计引物，进行 RT-PCR分析，发现农大603的肌醇-1-磷酸合成酶(Myo inositol-1-phosphate synthase , MIPS)基因的表达量高于徐薯18。采用3'RACE技术扩增出MIPS基因的3'末端cDNA。根据植物MIPS基因 5'端一段保守的氨基酸序列设计兼并引物，并与3'端的特异引物组合，扩增出该基因的5'端cDNA序列。DNA序列比对表明，甘薯MIPS基因与大豆(Glycine max)、番茄(Lycopersicon esculentum )的MIPS基因同源性较高，分别达83.63 %和83.89 %。甘薯MIPS基因全长cDNA的克隆，有利于进一步研究该基因与抗甘薯茎线虫病的关系。     

宇佐美曲霉木聚糖酶基因在大肠杆菌中的表达

在已知宇佐美曲霉E001菌株木聚糖酶Xyn II cDNA序列（Genbank登录号为DQ114485）的基础上，合成1对引物（含 EcoR I和Sal I酶切位点），以重组质粒pUCm-T-xyn II为模板，扩增得到编码Xyn II成熟肽的cDNA片段（555 bp）。将其与表达质粒pET-28a连接，构建了重组表达质粒pET-28a-xyn II，转化E. coli BL21-CodonPlus(DE3)-RIL，获得重组工程菌B21/xyn II。经IPTG诱导表达，木聚糖酶的比酶活力最高可达35.6 U/mg。最后采用金属Ni2+螯和层析柱对所表达的木聚糖酶进行纯化，达到了电泳纯。重组表达的木聚糖酶最适温度为45℃，最适pH值为4.6。     

人胰腺激肽释放酶cDNA的克隆、序列分析和原核表达

摘要: 为了研制人激肽释放酶（KLK1）特异单克隆抗体和进行重组酶的鉴定及纯化，根据已发表的人KLK1序列设计引物，用PCR扩增法从人胰腺单链cDNA库中特异地扩增出KLK1 cDNA。将其克隆入pGEM-T载体中,对5个重组质粒进行了序列测定，其中1个cDNA克隆的核苷酸序列与已发表的人肾/胰/唾液腺KLK1 cDNAs序列完全相同或有3个核苷酸差异。将去除信号肽序列的KLK1 cDNA以正确阅读框插入表达载体pGEX-4T-3,构建成重组质粒pGEX-KLK1。此重组质粒转化的E.coli 经IPTG诱导后表达分子量为48 kDGST-KLK1融合蛋白，表达产物主要以不溶性包涵体形式存在，可溶性部分能被Glutathione Sepharose 4B特异吸附，两者都能被GST特异单克隆抗体识别。用SDS-PAGE分离纯化的融合蛋白免疫小鼠，获得的抗血清的ELISA效价为1∶1600。结果表明，克隆的人KLK1 cDNA及其表达产物是正确的，可以用于人KLK1单克隆抗体的制备。     

猪粒细胞-巨噬细胞集落刺激因子（pGM-CSF)基因的克隆与原核表达

通过RT-PCR方法,从经ConA刺激的猪外周血淋巴细胞中扩增出猪粒细胞-巨噬细胞集落刺激因子（pGM-CSF）编码区的cDNA并将其克隆至pMD-19T载体,序列测定表明pGM-CSF基因的长度为435 bp,编码144个氨基酸。通过PCR方法获得缺失其N端17个氨基酸残基信号肽序列的成熟pGM-CSF蛋白基因,将其克隆至原核表达载体pET-32a（+）,构建重组表达质粒pET-GM并转入大肠杆菌(Escherichia coli )BL21（DE3）中进行诱导表达。经SDS-PAGE电泳和Western blot分析表明,表达的重组蛋白分子量约31 kD,主要以包涵体形式存在,表达量占菌体总蛋白的30.4% 。采用Ni2+-NTA亲和层析柱对经稀释复性的重组蛋白进行纯化,并采用MTT法以TF-1细胞检测纯化产物的生物学活性。结果表明,重组蛋白可有效刺激TF-1细胞增殖,其活性达到3.43×105 IU/mg。     

Isolation and characterization of 2S cocoa seed albumin storage polypeptide and the corresponding cDNA

The amine pool of cocoa is known to be an essential component for the development of the typical cocoa flavor. To better understand and to produce an intense in vitro cocoa flavor, identification of the polypeptides that are the source of the amine flavor precursor pool is essential. Chromatographic analysis of the polypeptide profile of unfermented cocoa resulted in identification of a novel storage polypeptide of M(r) 8515. The N-terminal sequence of the first 34 residues of the purified polypeptide shows similarity to 2S storage albumins of cotton and Brazil nut and sweet protein, Mabinlin. To identify the corresponding cDNA of the putative cocoa 2S albumin, 18 randomly chosen clones from the cDNA library of immature Theobroma cacao seed mRNA were sequenced, and a full-length cDNA clone encoding a protein harboring the N-terminal sequence of the novel polypeptide was selected. The open reading frame of the clone encodes a polypeptide of M(r) 17125. Comparison of the translated amino acid sequence of the precursor protein or the mature polypeptide against the Swiss-Prot and TrEMBL databases shows high sequence similarity (>52%) and identity (>38%) to many plant 2S albumins. Tryptic peptide mass fingerprinting of the purified polypeptide by high-performance liquid chromatography-electrospray ionization mass spectrometry shows 10 masses that match the expected tryptic peptides of the deduced sequence. Together with the published work on plant 2S albumin processing, the results presented here suggest that post-translational processing yields a 73-residue polypeptide (residue positions 78-150) corresponding to the 9 kDa subunit of the mature cocoa 2S albumin protein.     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.