Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Cloning, functional expression, and characterization of cystatin in sesame seed

A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.     

Effect of glycosylation modification (N-Q-(108)I --> N-Q-(108)T) on the freezing stability of recombinant chicken Cystatin overexpressed in Pichia pastoris X-33

The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence. The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.     

Method for bacterial expression and purification of sesame cystatin via artificial oil bodies

A method was developed for production of sesame cystatin, a thermostable cysteine protease inhibitor. Sesame cystatin was first expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a short hydrophilic linker peptide. Stable artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble oleosin-cystatin fusion protein. After centrifugation, the oleosin-cystatin fusion protein was exclusively found in the artificial oil bodies. Proteolytic cleavage with papain, a cysteine protease effectively inhibited by cystatin, separated soluble cystatin from oleosin that was firmly embedded in the artificial oil bodies. After recentrifugation, papain that coexisted with cystatin in the collected supernatant was denatured by incubating at 55 degrees C for 30 min. The insoluble denatured papain was removed by one more centrifugation, and the expressed cystatin of high yield and purity was harvested simply by concentrating the ultimate supernatant. Comparable inhibitory activity toward papain was observed between the expressed cystatin and the native one purified from sesame seeds. This method is presumably applicable to production of other protease inhibitors whose target proteases are economically available.     

Glycosylation modification improved the characteristics of recombinant chicken cystatin and its application on mackerel surimi

The recombinant and glycosylation chicken cystatins were expressed and secreted in the broth of Pichia pastoris X-33 transformant with apparent molecular masses (M) of 14 and 55 kDa, respectively. The glycosylation cystatin (glycocystatin) contained a polysaccharide chain that was composed of 50 DP of mannose residues. Because of the polymannosyl chain, the inhibitory ability in glycocystatin was 90.8% of recombinant cystatin. In addition to freeze-thawing stability, the thermal and pH stabilities as well as the susceptibility of glycocystatin were also enhanced. Both cystatins could improve the mackerel surimi gel by inhibiting the gel softening, which was derived from the hydrolysis of catheptic cysteine proteinases. Despite the additional amount of glycocystatin (8 units), twice that of recombinant cystatin, the 40 and 15% increases in breaking force and deformation of gels were also observed. Accordingly, the surimi gel was further improved by enhancing the stability of chicken cystatin.     

鸡破骨细胞分化因子（chRANKL）的克隆、表达和活性分析

为探讨RANKL以及OPG/RANKL/RANK通路在笼养蛋鸡骨质疏松发生中的作用,进行鸡破骨细胞分化因RANKL（receptor activator of NF-κB ligand）的克隆、表达并鉴定其活性。以成骨细胞总RNA为模板,利用RT-PCR和SOE-PCR技术体外扩增RANKL 基因,将PCR产物克隆至组氨酸标签的融合蛋白表达载体pET-32a(+),在异丙基－β－D硫代半乳糖苷（IPTG）诱导下,实现了chRANKL的有效表达,表达产物纯化后稀释成不同浓度梯度作用成熟破骨细胞观察其生物学活性。结果表明,凝胶电泳显示PCR扩增产物的长度为1200 bp左右,插入片段与Genebank上报道的鸡RANKL序列完全一致。重组表达载体转化BL21后经IPTG诱导获得大小约为64 Kd的重组蛋白,Western印迹表明重组蛋白具有抗原活性;并且纯化后的蛋白能刺激成熟破骨细胞,使骨吸收指数呈剂量依赖性增加,表明具有一定的活性。     

High-level production of recombinant chicken cystatin by Pichia pastoris and its application in mackerel surimi

A high level of the secreted form of recombinant chicken cystatin was expressed in Pichia pastoris X-33 by chromosomal integration of multiple copies of an expression cassette containing chicken cystatin under the control of glyceraldehyde-3-phosphate dehydrogenase promoter. The inhibition ability of the recombinant for papain-like proteinase was found to correspond to those of natural chicken cystatin. The recombinant cystatin substantially inhibited the proteolysis of myosin and gel softening, which consequently improved the gel properties of mackerel surimi.     

Preparation of Bioactive Recombinant Chicken Leptin Fusion Protein

将鸡Leptin成熟肽的cDNA片段插入表达载体pRSET A的Nhe Ⅰ和Hind Ⅲ两位点之间，构建重组表达质粒pLep -SCAU2并转化大肠杆菌(Escherichia coli )菌株BL21（DE3）, 将重组菌在含氨苄青霉素100 μg/mL 的LB培养基中培养至OD600大于0.6之后，经IPTG 0.05 mol/L诱导表达出分子量约为17.5 kD的含6×His标记的重组鸡Leptin，诱导4 h后表达量达最高，占总菌体蛋白的25％左右,且以包涵体形式存在。该重组鸡Leptin经50％Ni-NTA树脂纯化和透析复性折叠后分离出可溶性部分。对35日龄的矮脚黄鸡腹腔注射该可溶性重组鸡Leptin（2 mg/kg 体重）；注射后60 min内的采食量与对照组差异不显著 （P > 0.05），但在注射后80~120 min都显著低于对照组（P < 0.05）。Leptin处理公鸡的采食量在注射2 h后逐渐上升并在5.5 h时与对照组公鸡相同。但Leptin处理母鸡的采食量持续受到抑制，在注射后5.5 h时仍然显著低于对照组母鸡（P <0.01）。表明所表达的重组鸡Leptin能显著抑制鸡的采食量（母鸡比公鸡效果更为明显），证明所制备的重组鸡Leptin融合蛋白具有生物学活性。     

重组鸡β-防御素5 - β-防御素10双分子蛋白的构建及其理化特性分析

鸡抗菌肽属禽β-防御素（AvBD）类,是鸡先天性免疫的重要组成部分。研究将AvBD10基因定向插入到AvBD5-pGEX SalⅠ和NotⅠ双酶切位点上,构建了AvBD5-pGEX- AvBD10双基因共表达重组载体。将重组质粒转化大肠杆菌 (Escherichia coli ) BL21,于37 ℃不同时间进行诱导表达,SDS-PAGE检测外源基因的表达。结果表明,重组AvBD5-AvBD10双分子融合蛋白的分子量约为36 kD,重组双分子蛋白占菌体总蛋白的35%,重组菌表达产物以包涵体形式存在。重组双分子蛋白经纯化后,分别以对数生长中期的大肠杆菌[BL21(DE3-) 株]与致病性链球菌［Streptococcus（CAB株）］为检测菌,利用薄层平皿琼脂糖孔穴扩散法测定了重组双分子蛋白的抗菌活性,结果表明,重组双分子蛋白对这两种细菌都具有抗菌活性。并且对温度和pH有很高的稳定性,在-70～100 ℃或pH 3～12处理30 min仍具有抗菌活性。     

Lactobacillus reuteri PYR8亚油酸异构酶基因在大肠杆菌中的表达与活性检测

以Lactobacillus reuteri PYR8菌株基因组为模板,利用PCR方法扩增出亚油酸异构酶(linoleate isomerase,LI)基因,亚克隆到pET30a中构建原核表达载体pET30a-LI,并转化入大肠杆菌(Escherichia coli)BL21(DE3)中。其重组菌株在37℃条件下,经1.0mmol/L IPTG诱导表达重组LI蛋白,该蛋白以包涵体形式存在,表达量占菌体总蛋白的39.2%。采用Ni2 -NTA柱上复性法获得有活性的纯化重组亚油酸异构酶,其比活力5.12U/mg,是天然亚油酸异构酶比活力的2.5倍。     

猫白介素-18基因的分子克隆、序列分析及其表达研究

根据GenBank中报道的猫白介素-18基因（IL-18）序列，对用ConA刺激的实验猫外周血单核细胞（PBMCf）进行了RT-PCR扩增；将扩增得到的PCR产物纯化后克隆入pMD18-T中得到重组质粒pTIL-18，进行核苷酸序列测定，并与不同物种的IL-18基因进行了序列比较。结果该基因全长579bp，编码192个氨基酸。在推导的猫IL-18氨基酸序列中，无信号肽序列和潜在的N-联糖基化位点，但存在4个Cys残基。与不同物种IL-18相比，猫IL-18与犬、羊、牛和猪IL-18核苷酸和推导的氨基酸序列有较高的同源性，与小鼠和鸡IL-18有明显的种属差异。将pTIL-18双酶切，回收目的基因片段亚克隆到大肠杆菌表达载体pET28a中构建了重组质粒pETIL-18，转化大肠杆菌BL21（DE3），并用IPTG进行了诱导。结果重组菌菌体裂解物经SDS-PAGE电泳可检测到相对分子量为27.5 kDa的重组目的蛋白。经凝胶薄层扫描，目的蛋白表达量可占菌体蛋白的13.6%。     

Delta1-pyrroline-5-carboxylic acid formed by proline dehydrogenase from the Bacillus subtilis ssp. natto expressed in Escherichia coli as a precursor for 2-acetyl-1-pyrroline

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.     

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。     

耐碱锰超氧化物歧化酶（Mn-SOD）基因工程菌的构建及其表达条件研究

利用PCR技术以Bacillus sp.110-2基因组DNA为模板,扩增出606 bp编码锰超氧化物歧化酶的基因Mn-sodA,将其连接到原核表达载体pET-30a(+),得到重组载体pET-sodA,重组载体在大肠杆菌BL21(DE3)中经异丙基硫代-β-D-半乳糖苷（IPTG）诱导得到表达。SDS-PAGE分析显示融合表达产物的分子量大小为26.5k Da,同核酸序列测定的推导值相符。对含有sodA的基因工程菌表达情况研究表明：重组蛋白全部以可溶性形式存在;重组酶的比活力252 U/mg,为原始菌株的2.1倍,目的基因在大肠杆菌中实现了高效表达;而且,重组酶还保留了野生型酶显著的耐碱能力。BL21（pET-sodA）基因工程菌的构建一方面提供了一种可利用的耐碱蛋白资源,另一方面探索了一种极端酶的生产方法。     

Extracellular production of a functional soy cystatin by Bacillus subtilis

A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.     

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.     

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.     

Cloning and characterization of an antifungal class III chitinase from suspension-cultured bamboo ( Bambusa oldhamii ) cells

A class III chitinase cDNA (BoChi3-1) was cloned using a cDNA library from suspension-cultured bamboo ( Bambusa oldhamii ) cells and then transformed into yeast ( Pichia pastoris X-33) for expression. Two recombinant chitinases with molecular masses of 28.3 and 35.7 kDa, respectively, were purified from the yeast's culture broth to electrophoretic homogeneity using sequential ammonium sulfate fractionation, Phenyl-Sepharose hydrophobic interaction chromatography, and Con A-Sepharose chromatography steps. N-Terminal sequencing and immunoblotting revealed that both recombinant chitinases were encoded by BoChi3-1, whereas SDS-PAGE and glycoprotein staining showed that the 35.7 kDa isoform (35.7 kDa BoCHI3-1) was glycosylated and the 28.3 kDa isoform (28.3 kDa BoCHI3-1) was not. For hydrolysis of ethylene glycol chitin (EGC), the optimal pH values were 3 and 4 for 35.7 and 28.3 kDa BoCHI3-1, respectively; the optimal temperatures were 80 and 70 degrees C, and the K(m) values were 1.35 and 0.65 mg/mL. The purified 35.7 kDa BoCHI3-1 hydrolyzed EGC more efficiently than the 28.3 kDa isoform, as compared with their specific activity and activation energy. Both recombinant BoCHI3-1 isoforms showed antifungal activity against Scolecobasidium longiphorum and displayed remarkable thermal (up to 70 degrees C) and storage (up to a year at 4 degrees C) stabilities.     

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.