烟曲霉脯氨酰内肽酶在巴斯德毕赤酵母中的分泌表达与重组酶性质

【目的】研究烟曲霉脯氨酰内肽酶cDNA基因的异源表达及重组酶性质。【方法】以烟曲霉CICIM F0044总RNA为模板,反转录合成cDNA;再以cDNA为模板,通过PCR扩增去除自身信号肽的脯氨酰内肽酶基因,构建表达载体pPIC9K-PEP;电转化酵母宿主菌Pichia pastoris GS115,获得重组菌PEP-09;纯化并分析重组酶性质。【结果】重组菌摇瓶发酵酶活力最高可达647.3 U/L。表达产物纯化后的分子量为63 kD左右。重组酶最适反应温度为65°C,有较好的温度稳定性,在55°C保温8 h能保留90%以上的酶活力。该酶最适pH为5.5,在pH 3.0 9.0范围内有很好稳定性,在pH 6.0 8.0的缓冲液中37°C保温10 d酶活没有明显变化。【结论】烟曲霉脯氨酰内肽酶cDNA基因在巴斯德毕赤酵母中实现了分泌表达,重组酶活性稳定,有一定的应用潜力。     

重组内肽酶AspN的原核表达纯化和活性鉴定

蛋白内肽酶AspN是一种锌金属内切肽酶,能选择性切割天冬氨酸的N端肽键,广泛应用于蛋白的多肽制备及质量肽图谱鉴定。目前内肽酶AspN来源于细菌分泌,产量低,制备困难,成本高,极大地限制了该酶的应用。将脑膜脓毒性黄杆菌分泌的蛋白内肽酶Asp N所对应的基因克隆入表达载体p ET32a,导入E.coli BL21(DE3),首次运用原核表达系统进行可溶性融合表达,亲和层析对重组蛋白进行纯化。用HPLC、SDS-PAGE和荧光底物Anthranilyl-Ala-Phe-Ala-Phe-Asp-Val-Phe(NO2)-Tyr-Asp对重组酶进行酶活鉴定。结果表明重组的内肽酶Asp N具有与标准品基本一致的酶切活性,能够较好地应用于生物和制药领域。     

黑曲霉W3350脯氨酰内肽酶在大肠杆菌中的表达

目的：构建pET-PEP重组原核表达载体,并对表达产物进行鉴定。方法：采用RT-PCR技术从黑曲霉（Aspergillus niger）W3350中扩增出脯氨酰内肽酶（PEP）的基因,插入表达载体pET-22b（＋）,经EcoRⅠ和HindⅢ双酶切和DNA序列测定验证,将正确的重组质粒转入大肠杆菌BL21（DE3）中,IPTG（终浓度1mmol/L）诱导获得表达。结果：表达产物进行超声破碎,经SDS-PAGE电泳检测,PEP分子质量大约为57kDa,与理论值相符,通过酶活方法测定脯氨酰内肽酶酶活为0.656U/ml,约是出发菌株的5倍。结论：首次成功构建表达载体pET-PEP并在原核细胞中表达,为进一步研究PEP的生物学功能奠定了基础。     

AcSDKP对器官产纤维细胞功能的影响

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是广泛存在于哺乳动物体内的乙酰化小分子四肽,具有广泛的生理功能。近年来,AcSDKP参与组织器官细胞外基质沉积和纤维化调控的作用与机制越来越受到国内外学者的重视,已有研究发现其对心、肝、肺、肾等器官纤维化有抑制作用,而这些作用与各器官的产纤维细胞关系密切。本文就AcSDKP对心、肝、肺、肾内的产纤维细胞功能影响的研究进展展开综述。     

类赖氨酰氧化酶2与肿瘤转移和发生

类赖氨酰氧化酶2（lysyl oxidase—like 2,LOXL2）是赖氨酰氧化酶（1ysyl oxidase,LOX）基因家族的成员之一,其表达产物能促进胶原沉积。LOXL2的过表达能促进纤维化,并与肿瘤侵袭、转移及不良预后有关。目前大部分学者认为LOXL2是一种转移促进基因,也有实验支持其是一种肿瘤抑制基因。研究发现LOXL2可以通过激活Snail／Ecadherin通路或Src／FAK通路促进转移。LOXL2有望作为肿瘤生物标志物,用于预后判断,成为一个新的治疗靶点。     

AcSDKP对TGF-β1介导的大鼠心脏成纤维细胞MMP-1、MMP-2和MMP-9表达的调节

目的:探讨N-乙酰基-丝氨酰-天门冬酰-赖氨酰-脯氨酸(AcSDKP)对转化生长因子-β1(TGF-β1)诱导的大鼠心脏成纤维细胞MMP-1、MMP-2和MMP-9调节作用。方法:建立新生大鼠的心脏成纤维细胞系,分别用Westernblot法和明胶酶谱法检测心脏成纤维细胞MMP-1和MMP-2、MMP-9酶的表达。结果:10%血清能使心脏成纤维细胞MMP-2、MMP-9和MMP-1酶的表达增加,AcSDKP能进一步增加在10%血清诱导基础上三种酶的表达。TGF-β1促进心脏成纤维细胞MMP2和MMP-9酶的表达,而下调MMP-1酶表达。AcSDKP能进一步上调由TGF-β1诱导的心脏成纤维细胞MMP2和MMP-9酶的表达,并上调MMP-1酶表达。结论:AcSDKP对TGF-β1诱导的心脏成纤维细胞MMP-2、MMP-9和MMP-1酶表达有促进作用。     

串联亲和标签LOX蛋白慢病毒表达载体的构建及在乳腺癌细胞中的表达

目的:为了研究赖氨酰氧化酶(Lysyl Oxidase,LOX)及其相互作用蛋白质在乳腺癌中的功能,构建带StrepⅡ/FLAG串联亲和标签的重组LOX蛋白慢病毒表达载体并在乳腺癌细胞MDA-MB-231中表达。方法:设计引物通过聚合酶链式反应获得带StrepⅡ/FLAG串联亲和标签的LOX蛋白(LOX-SF)的亲本质粒,双酶切后鉴定测序克隆至GV303表达载体,连同慢病毒包装质粒共同转染293T得到GV303/LOX-SF慢病毒,将其转染MDA-MB-231细胞,使用荧光定量PCR和蛋白质印迹实验对细胞中重组蛋白LOX-SF进行检测。结果:通过串联亲和纯化获得LOX-SF重组蛋白,使用标签抗体成功鉴定到LOX-SF重组蛋白在MDA-MB-231细胞内稳定表达。结论:GV303/LOX-SF的构建,使带StrepⅡ/FLAG融合标签的LOX蛋白在MDA-MB-231中成功表达及纯化,为筛选和研究LOX及其相互作用蛋白在乳腺癌细胞内的功能奠定了实验基础。     

点状产气单胞菌脯氨酰内肽酶基因的克隆与表达

用活性筛选法从产气单胞菌点状亚种ST7833 (Aeromonas puctata subsp.puctata ST7833)的基因组中克隆了脯氨酰内肽酶 (Prolyl Endopeptidase,简称apPEP)的基因,测定了含有PEP基因的33kb DNA片段的序列,第202092bp编码了690个氨基酸组成的脯氨酰内肽酶,经检索是一种新的PEP基因。并构建了一株组成性高效表达PEP的基因工程菌BL21/pGEMPEP。BL21/pGEMPEP在 YH培养基中apPEP的表达量占菌体总蛋白的30%左右,活力是野生菌的112倍,表达产物主要为可溶性的胞内蛋白,约5%分泌到胞外。非还原SDSPAGE显示为单体,分子量为76kD,与基因序列预测的分子量一致。试管培养后纯化得到了纯度大于90%的重组脯氨酰内肽酶,比活力为67U/mg。     

AcSDKP对TGF-β1诱导大鼠MSCs向肌成纤维细胞分化的抑制作用

目的:研究N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartly-lysyl-proline,Ac SDKP)对转化生长因子β1(Transforming growth factor beta 1,TGF-β1)诱导大鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)向肌成纤维细胞(myofibroblast,MF)分化的影响,探讨Ac SDKP抗纤维化作用的可能机制。方法:全骨髓贴壁法分离培养大鼠骨髓MSCs。使用免疫组化,Western blotting技术分析α-SMA蛋白的表达以及Smad2/3,ERK1/2蛋白磷酸化的变化情况。结果:和对照组相比,TGF-β1诱导的MSC中α-SMA、磷酸化-Smad2/3及磷酸化-ERK1/2的表达大大增强,使用Ac SDKP干预细胞则三者的表达量明显下降且呈一定的剂量依赖性。结论:Ac SDKP可以显著抑制TGF-β1诱导的大鼠MSCs向MF分化,可能通过抑制TGF-β/Smad/ERK1/2信号通路的激活,从而发挥其抗器官纤维化作用。     

布氏锥虫苯丙氨酰-tRNA合成酶在大肠杆菌中的克隆、表达、纯化及活性测定

苯丙氨酰-tRNA合成酶是布氏锥虫蛋白合成过程中的一类重要酶,以其为靶点的抑制剂可能发展成为新一代的抗锥虫药物,但此前并没有分离锥虫苯丙氨酸-tRNA合成酶的报道。本研究用大肠杆菌成功克隆表达并纯化了布氏锥虫苯丙氨酰-tRNA合成酶并进行了活性测定。首先通过PCR方法从布氏锥虫细胞基因组中分别扩增出苯丙氨酰-tRNA合成酶的α亚基、β亚基的基因,依次克隆入pCOLADuet共表达载体,然后在大肠杆菌BL21(DE3)RIPL中进行了成功表达,并采用Ni-Bind亲和层析对其进行了纯化,最后用免疫印迹进行了鉴定。此外还采用放射性同位素方法进行了酶活性测定,这为下一步进行布氏锥虫苯丙氨酰-tRNA合成酶抑制物的设计和体外筛选奠定了良好的基础。     

A new procedure for enzymatic semisynthesis of human insulin by hydrolysis of single-chain des-(b-30)-lnsulin precursor with lysyl endopeptidase

It has been shown that the single-chain des-(B-30)-insulin precursor (SCI) can be converted into human insulin ester by transpeptidation using trypsin in the presence of a threonine derivative. The present study demonstrates that Achromobacter lyticus protease 1 (lysyl endopeptidase) can catalyze the transpeptidation reaction more efficiently than can trypsin. It is also shown that des-(B-30)-insulin (DAI) can be produced by hydrolysis of SCI with the lysyl endopeptidase. Since it is well known that SCI can be produced by gene technology, the following method is recommended for industrial production of human insulin ester: hydrolysis of SCI with lysyl endopeptidase followed by coupling of the resulting DAI with a threonine derivative using trypsin or lysyl endopeptidase.     

Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.     

Recombinant expression of hydroxylated human collagen in Escherichia coli

Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.     

A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum

Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness. This revised version was published online in August 2006 with corrections to the Cover Date.     

Medium for the production of Bacillus intermedius glutamyl endopeptidase by the recombinant Bacillus subtilis

A nutrient medium was elaborated for the efficient production of glutamyl endopeptidase by the recombinant Bacillus subtilis strain AJ73 bearing the Bacillus intermedius 3-19 glutamyl endopeptidase gene within a multicopy plasmid. Optimal concentrations of the main nutrients, peptone and inorganic phosphate, were found using a multifactor approach. To provide for active growth and efficient glutamyl endopeptidase production, the cultivation medium of the recombinant strain should be enriched in phosphorus, organic and inorganic nitrogen sources, and yeast extract. Complex protein substrates, such as casein and gelatin, enhanced the biosynthesis of glutamyl endopeptidase. At the same time, easily metabolizable carbon sources suppressed it. The production of glutamyl endopeptidase was stimulated by the bivalent cations Ca2+, Mg2+, and Co2+.     

Activation of Clostridium botulinum type B and E derivative toxins with lysine-specific proteases

Abstract Clostridium botulinum type B and E derivative toxins were activated with lysyl endopeptidase or endoproteinase Lys-C, which splits only the bond involving the carboxyl group of a lysine residue. Type B toxin was more efficiently activated with lysyl endopeptidase; type E toxin was more efficiently activated with trypsin. Type B toxin was split by the lysine-specific protease into 2 fragments of molecular sizes indistinguishable from those induced with trypsin. Type E toxin was split by the same protease into 3 fragments, 2 of which had M _r identical to those obtained with trypsin, the other having an M _r less than that of the heavy chain but greater than that of the light chain. These results attest that both activation and nicking of type B and E derivative toxins are ascribable to cleavage, not of an arginyl, but of a lysyl bond.     

Isolation and primary structure of the eclosion hormone of the tobacco hornworm, Manduca sexta

Eclosion hormone was isolated from trimmed pharate adult heads of Manduca sexta by an eight step purification procedure using a Heliothis virescens in vivo bioassay. The neuropeptide was active in second stadium M. sexta. The primary structure was determined by sequence analyses of the intact peptide and fragment peptides generated by lysyl endopeptidase, endoproteinase Glu-C, and proline-specific endopeptidase. The nature of the carboxyl terminus as a free acid was elucidated by analysis of amino acids from digestion of the intact peptide with lysyl endopeptidase, which liberated leucine, but no leucine amide. The complete primary structure of M. sexta closion hormone is H-Asn-Pro-Ala-Ile-Ala-Thr-Gly-Tyr-Asp-Pro-Met-Glu-Ile-Cys-Ile-Glu-Asn-Cy s-Ala- Gln-Cys-Lys-Lys-Met-Leu-Gly-Ala-Trp-Phe-Glu-Gly-Pro-Leu-Cys-Ala-Glu-Ser- Cys-Ile Lys-Phe-Lys-Gly-Lys-Leu-Ile-Pro-Glu-Cys-Glu-Asp-Phe-Ala-Ser-Ile-Ala-Pro- Phe-Leu-Asn-Lys-Leu-OH.     

Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli.

Chromatographic peptide mapping of lysyl endopeptidase digests of penicillin-binding protein 3 (PBP 3) of Escherichia coli revealed peptides that differed in retention time between the precursor and mature forms. The peptides were purified from a processing-defective (prc) mutant and a wild-type (prc+) strain. These peptides were identified as the C-terminal region of the precursor form and mature PBP 3 by amino acid sequencing. Each of the C-terminal peptides was cleaved into two fragments by trypsin digestion. By sequencing the resultant carboxyl-side fragment derived from the mature form, it was concluded that the C-terminal residue of mature PBP 3 was Val-577, and thus the Val-577-Ile-578 bond is the cleavage site for processing. This conclusion was consistent with the amino acid compositions of the relevant peptides, which suggested that the peptide from the cleavage site to the end of the deduced sequence (Ile-578-Ser-588) was present in the precursor but absent in the mature form. One lysyl peptide bond resisted both lysyl endopeptidase and trypsin and remained uncleaved in the peptide analyzed above.     

Identification of nucleotide binding sites in V-type Na+-ATPase from Enterococcus hirae

A and B subunits of the V-type Na+-ATPase from Enterococcus hirae were suggested to possess nucleotide binding sites (Murata, T. et al., J. Biochem., 132, 789-794 (2002)), although the B subunit did not have the consensus sequence for nucleotide binding. To further characterize the binding sites in the V-ATPase, we did the photoaffinity labeling study using 8-azido-[alpha-32P]ATP. A and B subunits were labeled with 8-azido-[alpha-32P]ATP when analysed with SDS polyacrylamide gel electrophoresis. The peptide fragment of A subunit obtained by lysyl endopeptidase digestion after labeling showed a molecular size of 9 kDa and its amino acid sequencing revealed that it corresponded to residues Arg423-Lys494. The peptide fragment from B subunit after photoaffinity labeling and lysyl endopeptidase digestion showed the size of 5 kDa and corresponded to residues Phe404-Lys443. In our structure model, these peptides were close to the adenine ring of ATP. We suggest that non-catalytic B subunit of E. hirae V-ATPase has a nucleotide binding site, similarly to eukaryotic V-ATPases and F-ATPases.