SARS病毒核衣壳蛋白的表达与鉴定

依据Genebank中SARS基因组序列和酵母菌对密码子的选择性,采用人工合成的方法,合成了优化的SARS病毒核衣壳蛋白(N)的全基因(1296bp),与CTL表位基因(195bp)重组后,将其克隆到酵母分泌型表达载体pPIC9K中,构建成重组表达载体pPIC9K-N.重组载体转化毕赤酵母GS115,并经MD平板和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115-pPIC9K-N.用甲醇诱导其分泌表达目的蛋白并对表达产物进行分析、浓缩与鉴定.结果表明,SARS病毒核衣壳蛋白能实现在毕赤酵母中高效表达,表达量达到20%,初步纯化后的产物具有良好的抗原特异性.     

Assembly and Immunogenicity of Human Papillomavirus Type 16 Major Capsid Protein（ HPV16 L1） in Pichia pastoris

IntroductionHPV16 L1 protein can self-assemblein vitrointovirus-like particles(VLPs),the morphology of which issimilar to that of native virions[1]. Human papilloma-viruses(HPV) belong to a family of nonenveloped,double-stranded DNA viruses, and more than…     

Optimization of high expression and purification of recombinant streptokinase and in vitro evaluation of its thrombolytic activity

BackgroundStreptokinase (SK) is a potent plasminogen activator naturally produced by beta-hemolytic streptococcus bacteria and used as a thrombolytic drug.ObjectivesOptimize high yield production of recombinant streptokinase (rSK) in Escherichia coli and evaluate its thrombolytic activity.MethodsSynthetic gene encoding mature SK protein with optimization for rare codons and mRNA secondary structure was cloned into the expression vector pET-3a and transformed into Escherichia coli BL21 (DE3). Seed banks were established for high rSK expression clones. The native rSK protein expression was optimized using IPTG induction. The nonsoluble rSK inclusion bodies were purified, denatured in 6 M guanidinium chloride, and refolded using the rapid dilution method. The refolded rSK protein was purified using anion exchange chromatography and evaluated with ELISA. The activity of rSK was evaluated using the casein digestion method and in vitro blood clot lysis assay with reference drug Sedonase as standard.ResultsSeed banks with high stable expression of native rSk (MW 47 kDa) were established. High rSK expression was optimized using 1 mM IPTG at bacterial OD600 0.6. The refolded rSK was prepared and purified successfully with high productivity (494 mg purified rsk/L culture). Using ELISA, the purified rSK molecular identity and conservation of native SK epitopes were confirmed. The enzymatic activity of the purified rSK was 1.945x10⁶ IU/mg with 62.94 ± 2.3% clot lysis efficiency.ConclusionA high yield production of proper rSK protein with in vitro thrombolytic activity similar to commercial SK has been achieved, suggesting a more cost-effective industrial production of its biosimilar drug.     

Structure of proteins in eukaryotic compartments

In-cell NMR in the yeast Pichia pastoris was used to study the influence of metabolic changes on protein structure and dynamics at atomic resolution. Induction of ubiquitin overexpression from the methanol induced AOX1 promoter results in the protein being localized in the cytosol and yields a well-resolved in-cell NMR spectrum. When P. pastoris is grown on a mixed carbon source containing both dextrose and methanol, ubiquitin is found in small storage vesicles distributed in the cytosol, and the resulting in-cell NMR spectrum is broadened. The sequestration of overexpressed proteins into storage vesicles, which are inaccessible to small molecules, was demonstrated for two unrelated proteins and two different strains of P. pastoris , suggesting its general nature.     

Constitutive Expression of a rhIL-2-HSA Fusion Protein in Pichia pastoris Using Glucose as Carbon Source

A constitutive expression vector for rhIL-2-HSA fusion protein production in yeast Pichia pastoris was constructed. The coding gene was placed in frame with the Saccharomyces cerevisiae α-factor secretion signal sequence under the control of the GAP promoter. The recombinant plasmid pGAPZαA-rhIL-2-HSA was integrated into the genome of the P. pastoris GS115. The effect of different carbon sources on rhIL-2-HSA fusion protein expression was evaluated in shaking flask cultures. We found that recombinant P. pastoris grew well and efficiently secreted rhIL-2-HSA fusion protein into the medium when using glucose as carbon source. To achieve higher production, the influence of initial pH and culture temperature was also evaluated. Fed-batch fermentation strategy using glucose as carbon source for constitutive expression of rhIL-2-HSA fusion protein was investigated in 5-L bioreactor and the expression level of rhIL-2-HSA could reach about 250 mg/L after 60-h fermentation. The rhIL-2-HSA fusion protein produced by this constitutive expression system was purified and exhibited a specific bioactivity of 1.040?×?10⁶ IU/mg in vitro. This study described constitutive expression of rhIL-2-HSA fusion protein by P. pastoris and development of a simple high-cell density fermentation strategy for biologically active rhIL-2-HSA fusion protein using glucose as sole carbon source.     

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.     

Enhanced alkaline protease production in addition to α-amylase via constructing a Bacillus subtilis strain

Bacillus subtilis Bios 11 strain was previously isolated and identified. This strain naturally produces a high level of α-amylase. The multicopy (pS1) plasmid that carries the complete alkaline protease aprA gene was introduced to this host strain by transformation. The newly constructed strain was found to express the aprA gene and produces a high level of alkaline protease. The level of α-amylase production was not affected compared with the parent strain. The pS1 plasmid in the new host was proved to be segregationally and structurally stable, and the multicopy aprA gene was expressed at the stationary phase. This expression did not affect growth rate and sporulation frequency. Moreover, the level of α-amylase was maintained. Both alkaline protease and α-amylase enzymes were purified using a single-step affinity chromatography column. The use of the newly constructed strain would be valuable to the enzyme industry and would promote recycling of some food-processing wastes.     

Asymmetric Baeyer-Villiger oxidations of 4-mono- and 4,4-disubstituted cyclohexanones by whole cells of engineered Escherichia coli.

Whole cells of an Escherichia coli strain that overexpresses Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase have been used for the Baeyer-Villiger oxidations of a variety of 4-mono- and 4,4-disubstituted cyclohexanones. In cases where comparisons were possible, this new biocatalytic reagent provided lactones with chemical yields and optical purities that were comparable to those obtained from the purified enzyme or a strain of bakers' yeast that expresses the same enzyme. The efficient production of cyclohexanone monooxygenase in the E. coli expression system (ca. 30% of total soluble protein) allowed these oxidations to reach completion in approximately half the time required for the engineered bakers' yeast strain. Surprisingly, 4,4-disubstituted cyclohexanones were also accepted by the enzyme, and the enantioselectivities of these oxidations could be rationalized by considering the conformational energies of bound substrates along with the enzyme's intrinsic enantioselectivity. The enzyme expressed in E. coli cells also oxidized several 4-substituted cyclohexanones bearing polar substituents, often with high enantioselectivities. In the case of 4-iodocyclohexanone, the lactone was obtained in > 98% ee and its absolute configuration was assigned by X-ray crystallography. The crystal belongs to the monoclinic P2(1) space group with a = 5.7400(10), b = 6.1650(10), c = 11.377(2) A, b = 99.98(2) degrees, and Z = 2. Taken together, these results demonstrate the utility of an engineered bacterial strain in delivering useful chiral building blocks in an experimentally simple manner.     

来源于类芽孢杆菌属新型耐有机溶剂脂肪酶的克隆、表达与性质研究（英文）

脂肪酶(EC 3.1.1.3)全称为三酰基甘油水解酶,是一类能够将长链脂肪酸甘油酯水解成脂肪酸和二甘酯、单甘酯或甘油的酯键水解酶.它除了能够水解脂肪外,还具有催化酯化反应、酯交换反应、酸解反应、醇解反应以及氨解等反应的性质.在脂肪酶催化的反应中,通常用有机溶剂代替水.有机溶剂可以转移合成反应的平衡方向,通过溶剂工程修饰酶的选择性能够提高底物的溶解度、有机相产物的回收率、酶的热稳定性.但有机溶剂对酶活性和稳定性有不同程度的影响.因此,寻找在有机溶剂中表现出高活性和稳定性的脂肪酶是一个亟待解决的重要课题.由于微生物种类多、作用底物专一性强,且微生物来源的脂肪酶一般分泌到胞外,因此微生物脂肪酶是工业用脂肪酶的重要来源.目前,微生物脂肪酶的研究主要集中于根霉属(Rhizopus)、曲霉属(Aspergillus)、青霉属(Penicillium)、毛霉属(Mucor)、地霉属(Geotrichum)、假丝酵母属(Candida)、假单胞菌属(Pseudomonas)、伯克霍尔德菌属(Burkholderia)等具有工业应用价值的菌株.很少有类芽孢杆菌属所产脂肪酶进行相关酶学性质的研究.我们以Paenibacillus pasadenensis CS0611为出发菌株,在全基因序列草图中得到了一个新型脂肪酶基因lp2252.以Paenibacillus pasadenensis CS0611基因组为模板,设计特异性引物对目标序列进行扩增,并成功将其插入到表达载体p ET-28a中得到含有目的基因的重组质粒.在E.coli BL21(DE3)中,脂肪酶lp2252经0.1mmol/L的IPTG诱导后在20°C实现了高水平表达.重组脂肪酶的活性约为野生型的1631倍.用镍离子亲和层析柱快速、高效地纯化了两端带有组氨酸标签的重组脂肪酶,回收率为63.5%,纯化因子为10.78.纯化后的脂肪酶最适温度为50°C,在20-40°C范围内具有良好的稳定性.最适pH值为7,属于中性脂肪酶,同时在pH 3.0-8.0间具有较高稳定性.在金属离子如钙、镁离子和一些非离子表面活性剂的作用下,其活性有所提高.此外,纯化后的脂肪酶可被一系列水溶性有机溶剂激活,例如一些短链醇.而对某些水不溶性有机溶剂,其也具有高度的耐受性.综上所述,本文所涉新型脂肪酶在非水相催化领域具有广泛的应用和前景.     

Engineering of a Pichia pastoris Expression System for High-Level Secretion of HSA/GH Fusion Protein

Human serum albumin (HSA) and human growth hormone (hGH) fusion protein [HSA/GH] is a promising long-acting form of GH to treat GH deficiency. This study attempted to engineer a P. pastoris strain for high-level production of HSA/GH to be used in basic research and clinical application. Strains contained two, three, and seven copies of HSA/GH gene were screened by selecting against Zeocin resistance. The results revealed that introducing two to three copies of HSA/GH gene was sufficient to give a significant increase in secretion level, compared with one copy of HSA/GH gene. No significant differences were observed between two to three copies and seven copies. Co-expression with either one copy of exogenous ERO1 or PDI in a strain carrying multicopies of HSA/GH gene led to varying degrees of increase in HSA/GH secretion. The effect of introducing multicopies of PDI was similar to that of one copy of PDI, but introducing excess copies of ERO1 reduced HSA/GH secretion. Simultaneous co-expression with PDI and ERO1 was less effect than either PDI co-expression or ERO1 co-expression. A strain showing higher secretion level was successfully applied to large-scale fermentation with the productivity of 3–4 g/l.     

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv–GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv–GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv–GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.     

Large-scale Generation of Affinity-purified Recombinant Phytochrome Chromopeptide

Abstract— Two different yeast expression systems, Pichia pastoris and Hansenula polymorpha, are compared for their capability to express in functional form the 65 kDa N-ter-minal portion of oat phytochrome A (phyA, spanning amino acids 1-595). The front half of phytochrome was selected for this investigation because it exhibits a greater stability than the full-length protein, and it harbors full spectroscopic and kinetic properties of phytochrome, allowing an exact proof of the functional integrity of the recombinant material. In the comparison between the two expression systems used, special emphasis was given to optimizing the yield of the expression and to improving the quality of the expressed material with respect to the proportion of functional protein. From identical volumes of cell culture, H. polymorpha synthesized between 8- and 10-fold more functional protein than P. pastoris. Following the observation by Wu and Lagarias (Proc. Natl. Acad. Sci. USA 93, 8989-8994,1996) that P. pastoris endogenously produces the chromophore of phytochrome, phytochromobilin (PpHB) in significant amounts that leads to formation of spectrally active phytochrome during expression, the invention of an alternative high-yield expression system was strongly demanded. A Histag was attached to the C-terminus of the recombinant protein, which allows for a convenient and efficient purification and selects the full-length proteins over translationally truncated peptides. Fully reconstituted chromo-proteins showed an A660A280 ratio of 1.2, indicating the high degree of reconstitutable apoprotein obtained by this procedure. The assembly between apoprotein and the chromophore phycocyanobilin when followed time-resolved yielded a time constant (obs) of 35 s. The λ_max values of the red-(Pr) and the far red-absorbing (Pfr) forms of phytochrome (665 and 729 nm) of the recombinant 65 kDa chromopeptide, reconstituted with PcjiB are nearly identical to those of native full-length oat phytochrome. The kinetic parameters of the affinity-purified 65 kDa phytochrome chromoprotein for the Pr I700 Pfr conversion are compared to those of the recombinant 65 kDa chromoprotein, lacking the His-tag and to wild-type oat phytochrome. Referring to wild-type phytochrome allows determination of whether the recombinant material has lost spectral properties during the purification procedure. The decay of the primary intermediate (I₇₀₀) occurs with nearly the same time constant for the His-tagged chromoprotein and for the reference (110 and 90 mUs, respectively). The formation of the Pfr form was fitted with three exponentials in both the His-tagged and the reference chromoprotein with the middle component being slightly smaller and the longest component being remarkably larger for the His-tagged protein (1.5, 10 and 300 ms) than for the reference (1.4, 18 and 96 ms). This selective slowing down of the long kinetic component in the millisecond time range may be indicative of stronger interactions between protein domains involving the C-terminus that in the His-tagged form exhibits increased polarity.     

Expression and Secretion of the Human Erythropoietin Using an Optimized cbh1 Promoter and the Native CBH I Signal Sequence in the Industrial Fungus Trichoderma reesei

The human erythropoietin (HuEPO) structural gene was fused with the secretion signal of the Trichoderma reesei cellobiohydrolase I and controlled by a newly optimized cbh1 promoter in an integrated expression vector pTrCBH-EPO. The recombinant HuEPO construct was transformed into two different T. reesei strains, a protease-deficient strain RutC-30 M3, and a glycosylation-modified strain T108. After lactose induction, the heterologous rHuEPO was found to be stably expressed in the selected transformants T47 (derived from RutC-30 M3) and T112 (derived from T108), which were shown to have high genetic stability. Secretion of erythropoietin in these transformants was further confirmed by SDS-PAGE and western blot analysis. Moreover, the secreted rHuEPO from T112 had an apparent molecular weight of 32 kDa, which was higher than from T47 (28 kDa) and similar to that of mammals (more than 30 kDa). These results demonstrate the potential of using industrial filamentous fungi for the production of human-derived erythropoietin.     

Purification and Kinetic Properties of Human Recombinant Dihydrofolate Reductase Produced in <Emphasis Type="Italic">Bombyx mori</Emphasis> Chrysalides

Recent reports describe the inhibition of human dihydrofolate reductase (hDHFR) by natural tea polyphenols. This finding could explain the epidemiologic data on their prophylactic effects for certain forms of cancer, and it raises the possibility that natural and synthetic polyphenols could be used in cancer chemotherapy. In order to obtain larger quantities of hDHFR to support structural studies, we established and validated a baculovirus system for the expression of this protein in Bombyx mori chrysalides (pupae of the silkworm enclosed in a cocoon). To isolate the expressed protein, whole infected pupae were homogenized, and the expressed protein was purified by affinity chromatography. Here, we demonstrate the efficient expression of recombinant hDHFR in this model and report that this newly expressed protein has high enzymatic activity and kinetic properties similar to those previously reported for recombinant hDHFR expressed in Escherichia coli. The purified protein showed dissociation constants for the binding of natural polyphenols similar to that expressed in E. coli, which ensures its usage as a new tool for further structural studies. Although the hDHFR yield per individual was found to be lower in the chrysalides than in the larvae of B. mori, the former system was optimized as a model for the scaled-up production of recombinant proteins. Expression of proteins in chrysalides (instead of larvae) could offer important advantages from both economic and biosecurity aspects.     

Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression

Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.     

Pentamethylquercetin improves adiponectin expression in differentiated 3T3-L1 cells via a mechanism that implicates PPARγ together with TNF-α and IL-6

Adiponectin is an adipocyte-derived hormone that plays a pivotal role in the regulation of lipid and glucose metabolism. Up-regulation of adiponectin expression and production has been shown to benefit for metabolic disorders, including type 2 diabetes, hyperlipidemia, etc. The present study investigated whether the novel polymethoxylated flavonoid pentamethylquercetin (PMQ), a member of polymethoxylated flavonoids family which is present in seabuckthorn (Hippophae L.) would affect adiponectin production in differentiated 3T3-L1 adipocytes. It was found that PMQ increased the adiponectin mRNA and protein expressions in adipocytes in time- and concentration-dependent manners. The PPARγ pathway plays a important roles in this effect of PMQ because blockade of PPARγ by GW9662 eliminates the PMQ-induced up-regulation of adiponectin expression. Furthermore, significant decreases of mRNA expression and secretion of TNF-α and IL-6 were also observed in PMQ-treated cells. Taken together, our study demonstrated that PMQ up-regulates adiponectin expression via a mechanism that implicates PPARγ together with TNF-α and IL-6, suggesting that PMQ might be a potential candidate for the treatment of metabolic diseases.     

棘孢曲霉β-葡萄糖苷酶Ⅰ在毕赤酵母中的表达及烷基糖苷的催化合成

将来自棘孢曲霉(Aspergillus aculeatus NO. F-50)的β-葡萄糖苷酶Ⅰ在毕赤酵母中分泌表达. 初步研究表明, 目的蛋白得到较好表达, 以对硝基酚-β-D-葡萄糖苷(4-Nitrophenyl-β-D-glucopyranoside, pNPG)为底物, 重组β-葡萄糖苷酶Ⅰ酶促反应的最适温度为65 ℃, 最适pH为5.0, 50 ℃下反应发酵上清液中的酶活力可达33.8 U/mL, 蛋白表达量最高可达0.388 mg/mL. 该重组酶可通过逆水解或转糖苷反应催化合成烷基糖苷. 在有机-水双相反应体系中, 初步优化了pH 值、 含水量、 葡萄糖浓度及酶量等条件. 结果表明, 在优化的反应条件下, 丁基、 己基、 辛基和癸基葡萄糖苷最大产率分别为51.4%, 28.8%, 6.9%和3.0%.     

Production, purification, and characterization of a polygalacturonase from a new strain of kluyveromyces marxianus isolated from coffee wet-processing wastewater

A new high polygalacturonase (PG)-producing Kluyveromyces marxianus strain was isolated from coffee wet-processing wastewater. PG production in this strain is not repressed in the presence of 100g/L of glucose and, being growth-associated, reached its maximum accumulation in the culture medium at the beginning of the stationary phase. Oxygen and galacturonic acid negatively regulated enzyme synthesis, and glucose as the carbon source afforded better enzyme yields than lactose. The data reported here show that this strain exhibits the highest index of PG production among the wild-type strains reported so far (18.8U/mL). PG was readily purified by ion-exchange chromatography on SP-Sepharose FF. The activity corresponded to a single protein with an M _r of 41.7 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was stable in the pH range of 3.0–5.0 and displayed an optimal temperature of 55°C; it showed a typical endo-splitting way of substrate hydrolysis and exhibited a fair degree of activity on pectin with a high degree of esterification.     

人三叶因子3双结构域突变体的构建

利用基因工程方法在大肠杆菌-酶母穿梭质粒pPIC9K上构建了人三叶因子3(HumanTrefoilFactor3,hTFF3)双结构域突奕体基因,采用毕氏酵母表达系统对目的基因进行了分泌表达。经S-Sepharose,Q-Sepharose,SephacrylS-100纯化后得到突变体蛋白,主要以单体形式存在。SDS-PAGE表明重组蛋白分子量约为12000,并在Westernblotting印迹实验中证明能被抗hTFF3抗体所识别,N-端氨基酸测序与天然hTFF3-一致,质谱检测纯化的蛋白出现单体和双体两种形式。用重组蛋白对乙醇诱导的大鼠胃溃疡进行实验治疗表明hTFF3双体突变体与天然形成的双体蛋白具有相同的活力,并且活力高于单体形式的hTFF3。