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1.
提高Xa因子酶切效率的策略 总被引:1,自引:0,他引:1
为提高Xa因子对融合蛋白CBD-IGF和CBD-PACAP的酶切效率 ,以便高效释放非融合的重组多肽 ,利用基因工程技术在两个融合蛋白中Xa因子识别位点 (Ile-Glu-Gly-Arg↓ )前均引入 7个氨基酸组成的富含甘氨酸柔性短肽 (Gly-Thr-Gly-Gly-Gly-Ser-Gly)。纤维素亲和层析纯化各个融合蛋白 ,比较Xa因子对引入短肽前、后融合蛋白的酶切效率。比较结果表明 :短肽的引入不同程度地提高了融合蛋白CBD-IGF和CBD-PACAP对Xa因子的敏感性 ;但总体上CBD-IGF对Xa因子的敏感性比CBD-PACAP低。此研究结果提供了一种提高Xa因子酶切效率的策略。 相似文献
2.
含有Fxa切割位点的抗菌肽X在大肠杆菌中的融合表达 总被引:3,自引:0,他引:3
抗菌肽是昆虫体液免疫的重要成分[1,2 ] ,它们的分子量较小 ,具有抗菌、抗病毒和杀伤某些肿瘤细胞的功能 ,而不破坏人体正常细胞。基于它的这种选择性效应和分子小、无抗原性的特点 ,可望成为新一代的抗菌、抗肿瘤药物。然而 ,天然抗菌肽来源十分困难 ,不能满足研究和临床应用的需要 ,通过基因工程技术生产抗菌肽已成为人们普遍关注的焦点。抗菌肽CMIV是从家蚕蛹中分离并测定了其一级结构的新型抗菌肽 ,它由 35个氨基酸组成 ,不含甲硫氨酸 ,C 末端为酰胺[3 ] 。抗菌肽X是中国家蚕抗菌肽CMIV的变体 ,其一级结构与天然的抗菌肽CM… 相似文献
3.
A Computer Modeling Study of the Interaction Between Tissue Factor Pathway Inhibitor and Blood Coagulation Factor Xa 总被引:1,自引:0,他引:1
Activation of blood coagulation factor X to factor Xa (FXa) is inhibited by tissue factor pathway inhibitor (TFPI). The second Kunitz-type inhibitory domain (K2) of TFPI binds a catalytic domain of FXa, whereas the first domain (K1) does not. We analyzed computer models of complexes of FXa with K1 or K2, which were made using a crystal structure of FXa. Favorable hydrophobic interaction was observed in the complex of FXa with K2. Furthermore, we constructed a tertiary structure of FXa using CHIMERA to assess the accuracy of a homology modeling method. The isolated model structure of FXa agreed well with the crystal structure, but analyses of complexes of this structure with K1 or K2 revealed that the models of complexes could not provide clear evidence of greater binding ability to K2 because of the positional difference of a few side chains interacting with the inhibitor. 相似文献
4.
Mochizuki A Nagata T Kanno H Takano D Kishida M Suzuki M Ohta T 《Bioorganic & medicinal chemistry letters》2011,21(24):7337-7343
We have optimized 2-aminomethylphenylamine derivative as a factor Xa inhibitor. Several polar functional groups were introduced in the central phenyl ring, and we focused on zwitter ionic compound showing continuous inhibitory activity in oral administration test. In vitro and oral activities were improved by optimization of S1 and S4 ligands. Incorporating the interaction with S1-β pocket enhanced in vitro factor Xa inhibitory activity to less than 1 nM. Many zwitter ionic compounds showed long duration of action and potent inhibitory activity and high AUC values in oral administration tests to monkeys. 相似文献
5.
The influence of linker design on fusion protein production and performance was evaluated when a family 9 carbohydrate-binding module (CBM9) serves as the affinity tag for recombinant proteins expressed in Escherichia coli. Two bioinformatic strategies for linker design were applied: the first identifies naturally occurring linkers within the proteome of the host organism, the second involves screening peptidases and their known specificities using the bioinformatics software MEROPS to design an artificial linker resistant to proteolysis within the host. Linkers designed using these strategies were compared against traditional poly-glycine linkers. Although widely used, glycine-rich linkers were found by tandem MS data to be susceptible to hydrolysis by E. coli peptidases. The natural (PT)(x)P and MEROPS-designed S(3)N(10) linkers were significantly more stable, indicating both strategies provide a useful approach to linker design. Factor X(a) processing of the fusion proteins depended strongly on linker chemistry, with poly(G) and S(3)N(10) linkers showing the fastest cleavage rates. Luminescence resonance energy transfer studies, used to measure average distance of separation between GFP and Tb(III) bound to a strong calcium-binding site of CBM9, revealed that, for a given linker chemistry, the separation distance increases with increasing linker length. This increase was particularly large for poly(G) linkers, suggesting that this linker chemistry adopts a hydrated, extended configuration that makes it particularly susceptible to proteolysis. Differential scanning calorimetry studies on the PT linker series showed that fusion of CBM9 to GFP did not alter the T(m) of GFP but did result in a destabilization, as seen by both a decrease in T(m) and DeltaH(cal), of CBM9. The degree of destabilization increased with decreasing length of the (PT)(x)P linker such that DeltaT(m) = -8.4 degrees C for the single P linker. 相似文献
6.
Corte JR Fang T Pinto DJ Han W Hu Z Jiang XJ Li YL Gauuan JF Hadden M Orton D Rendina AR Luettgen JM Wong PC He K Morin PE Chang CH Cheney DL Knabb RM Wexler RR Lam PY 《Bioorganic & medicinal chemistry letters》2008,18(9):2845-2849
Introduction of the phenyl piperidinone and phenyl pyridinone P4 moieties in the anthranilamide scaffold led to potent, selective, and orally bioavailable inhibitors of factor Xa. Anthranilamide 28 displayed comparable efficacy to apixaban in the rabbit arteriovenous-shunt (AV) thrombosis model. 相似文献
7.
Hua Ling 《Biologia》2007,62(2):119-123
For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are
capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products
to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion
technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this
article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with
progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals
at large scale. 相似文献
8.
The cleavage of poliovirus precursor polypeptides occurs at specific amino acid pairs that are recognized by viral proteinases. Most of the polio-specific cleavages occur at glutamine-glycine (Q-G) pairs that are recognized by the viral-encoded proteinase 3C (formerly called P3-7c). In order to carry out a defined molecular genetic study of the enzymatic activity of protein 3C, we have made cDNA clones of the poliovirus genome. The cDNA region corresponding to protein 3C was inserted into an inducible bacterial expression vector. This recombinant plasmid (called pIN-III-C3-7c) utilizes the bacterial lipoprotein promoter to direct the synthesis of a precursor polypeptide that contains the amino acid sequence of protein 3C as well as the amino- and carboxy-terminal Q-G cleavage signals. These signals have been previously shown to allow autocatalytic production of protein 3C in bacteria transformed with plasmid pIN-III-C3-7c. We have taken advantage of the autocatalytic cleavage of 3C in a bacterial expression system to study the effects of site-specific mutagenesis on its proteolytic activity. One mutation that we have introduced into the cDNA region encoding 3C is a single amino acid insertion near the carboxy-terminal Q-G cleavage site. The mutant recombinant plasmid (designated pIN-III-C3-mu 10) directs the synthesis of a bacterial-polio precursor polypeptide that is like the wild-type construct (pIN-III-C3-7c). However, unlike the wild-type precursor, the mutant precursor cannot undergo autocatalytic cleavage to generate the mature proteinase 3C. Rather, the precursor is able to carry out cleavage at the amino-terminal Q-G site but not at the carboxy-terminal site. Thus, we have generated an altered poliovirus proteinase that is still able to carry out at least part of its cleavage activities but is unable to be a suitable substrate for self-cleavage at its carboxy-terminal Q-G pair. 相似文献
9.
Franciskovich JB Masters JJ Weber WW Klimkowski VJ Chouinard M Sipes PR Johnson LM Snyder DW Chastain MK Craft TJ Towner RD Gifford-Moore DS Froelich LL Smallwood JK Foster RS Smith GF Liebeschuetz JW Murray CW Young SC 《Bioorganic & medicinal chemistry letters》2007,17(24):6910-6913
Several P4 domain derivatives of the general d-phenylglycinamide-based scaffold (2) were synthesized and evaluated for their ability to bind to the serine protease factor Xa. Some of the more potent compounds were evaluated for their anticoagulant effects in vitro. A select subset containing various P1 indole constructs was further evaluated for their pharmacokinetic properties after oral administration to rats. 相似文献
10.
Frederick J. Walker Charles T. Esmon 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(3):405-415
The effects of heparin on prothrombin activation have been examined. Heparin was found to inhibit the rate of prothrombin activation by Factor Xa, calcium and phospholipid. In the absence of phospholipid, heparin had no effect on the rate of prothrombin activation. In contrast, heparin was found to increase the rate of activation of prethrombin-1 and prethrombin-2. Initial velocity studies indicated that heparin blocks lipid stimulation of prothrombin activation. In accord with this, binding studies demonstrated that heparin could displace Factor Xa, and in separate experiments, prothrombin, from phospholipid vesicles. 相似文献
11.
水稻白叶枯病是水稻生产上的主要细菌病害之一。从野生稻中发掘优异的水稻白叶枯病抗性材料,可以拓宽栽培稻抗白叶枯病遗传基础。经过温室接菌鉴定和PCR标记分析,对云南野生稻进行Xa21基因的检测鉴定。温室接菌鉴定表明,云南野生稻对广谱致病小种PX099及云南强致病菌Y8具有较好的抗性能力,特别是疣粒野生稻对致病菌株达到免疫程度;PCR标记分析表明,云南野生稻不含有Xa21基因,但含有与Xa21基因某些区域同源的片段。本研究结果为寻找新的抗源材料及快速发掘利用云南野生稻中的抗白叶枯病基因提供理论依据。 相似文献
12.
A semisynthesis of two ent-strobane diterpenoids strobols C (7) and D (14) was accomplished via a Wagnar-Meerwein rearrangement. Compounds 7, 14, and the intermediate products were evaluated for their inhibition on factor Xa (FXa). Among all the compounds screened for FXa inhibitory activity, three compounds 6, 7, and 9 showed significant inhibitory activities with IC50 values of 1067?±?164, 81?±?11, 1023?±?89?nM, respectively. The inhibitory activity on FXa described in this study highlight the importance of structural modification based on natural products in the development of FXa inhibitors. 相似文献
13.
Protein flexibility and conformational diversity is well known to be a key characteristic of the function of many proteins. Human blood coagulation proteins have multiple substrates, and various protein-protein interactions are required for the smooth functioning of the coagulation cascade to maintain blood hemostasis. To address how a protein may cope with multiple interactions with its structurally diverse substrates and the accompanied structural changes that may drive these changes, we studied human Factor X. We employed 20 ns of molecular dynamics (MD) and steered molecular dynamics (SMD) simulations on two different conformational forms of Factor X, open and closed, and observed an interchangeable conformational transition from one to another. This work also demonstrates the roles of various aromatic residues involved in aromatic-aromatic interactions, which make this dynamic transition possible. 相似文献
14.
Mahmoud A. Ibrahim Abdel-Hady M. Ghazy Tahany M. Maharem Mohamed I. Khalil 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2001,130(4)
An inhibitor of factor Xa (FXa) was isolated from the nymphs of the camel tick Hyalomma dromedarii by a combination of chromatography on DEAE-cellulose and Sephacryl S-300 columns. The isolated nymphal FXa inhibitor turned out to be a homogenous preparation of a single polypeptide chain (15 kDa) as judged by both the native and denatured SDS-PAGE. Its pI value ranged from 7.7 to 7.9. The inhibitor is a potent anticoagulant since it prolonged both the activated partial thromboplastin time (APTT) and the prothrombin time (PT) of the camel plasma in a concentration-dependent manner. Its activity was threefold lower toward thrombin than FXa, but it did not inhibit any of the proteases; trypsin, α-chymotrypsin, papain, pepsin and subtilisin. The inhibitor binds at two sites on FXa uncompetitively with an inhibition constant (Ki) value of 134 nM. 相似文献
15.
Yamamoto H Ida T Tsutsuki H Mori M Matsumoto T Kohda T Mukamoto M Goshima N Kozaki S Ihara H 《Microbiology and immunology》2012,56(4):245-253
The botulinum neurotoxin light chain (BoNT-LC) is a zinc-dependent metalloprotease that cleaves neuronal SNARE proteins such as SNAP-25, VAMP2, and Syntaxin1. This cleavage interferes with the neurotransmitter release of peripheral neurons and results in flaccid paralysis. SNAP, VAMP, and Syntaxin are representative of large families of proteins that mediate most membrane fusion reactions, as well as both neuronal and non-neuronal exocytotic events in eukaryotic cells. Neuron-specific SNARE proteins, which are target substrates of BoNT, have been well studied; however, it is unclear whether other SNARE proteins are also proteolyzed by BoNT. Herein, we define the substrate specificity of BoNT-LC/B, /D, and /F towards recombinant human VAMP family proteins. We demonstrate that LC/B, /D, and /F are able to cleave VAMP1, 2, and 3, but no other VAMP family proteins. Kinetic analysis revealed that all LC have higher affinity and catalytic activity for the non-neuronal SNARE isoform VAMP3 than for the neuronal VAMP1 and 2 isoforms. LC/D in particular exhibited extremely low catalytic activity towards VAMP1 relative to other interactions, which we determined through point mutation analysis to be a result of the Ile present at residue 48 of VAMP1. We also identified the VAMP3 cleavage sites to be at the Gln 59-Phe 60 (LC/B), Lys 42-Leu 43 (LC/D), and Gln 41-Lys 42 (LC/F) peptide bonds, which correspond to those of VAMP1 or 2. Understanding the substrate specificity and kinetic characteristics of BoNT towards human SNARE proteins may aid in the development of novel therapeutic uses for BoNT. 相似文献
16.
Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins 总被引:2,自引:0,他引:2
Dian C Eshaghi S Urbig T McSweeney S Heijbel A Salbert G Birse D 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2002,769(1):133-144
In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity. 相似文献
17.
Yoshikawa K Yoshino T Yokomizo Y Uoto K Naito H Kawakami K Mochizuki A Nagata T Suzuki M Kanno H Takemura M Ohta T 《Bioorganic & medicinal chemistry letters》2011,21(7):2133-2140
We previously reported on a series of cyclohexanediamine derivatives as highly potent factor Xa inhibitors. Herein, we describe the modification of the spacer moiety to discover an alternative scaffold. Ethylenediamine derivatives possessing a substituent at the C1 position in S configuration and phenylenediamine derivatives possessing a substituent at the C5 position demonstrated moderate to strong anti-fXa activity. Further SAR studies led to the identification of compound 30h which showed both good in vitro activity (fXa IC50 = 2.2 nM, PTCT2 = 3.9 μM) and in vivo antithrombotic efficacy. 相似文献
18.
Greenwood JM Gilkes NR Miller RC Kilburn DG Warren RA 《Biotechnology and bioengineering》1994,44(11):1295-1305
Fusion of the leader peptide and the cellulose-binding domain (CBD) of endoglucanase A (CenA) from Cellulomonas fimi, with of without linker sequences, to the N-terminus of alkaline phosphatase (PhoA) from Escherichia coli leads to the accumulation of significant amounts of the CBD-PhoA fusion proteins in the supernatants of E. coli cultures. The fusion proteins can be purified from the supernatants by affinity chromatography on cellulose. The fusion protein can be desorbed from the cellulose with water or guanidine-HCl. If the sequence IEGR in present between the CBD and PhoA, the CBD can be cleaved from the PhoA with factor Xa. The efficiency of hydrolysis by factor Xa is strongly in fluenced by the amino acids on either side of the IEGR sequence. The CBD released by factor Xa is removed by adsorption to cellulose. A nonspecific proteases from C. fimi, which hydrolyzes native CenA between the CBD and the catalytic domain, may be useful for removing the CBD from some fusion proteins. (c) 1994 John Wiley & Sons, Inc. 相似文献
19.
Smyth DR Mrozkiewicz MK McGrath WJ Listwan P Kobe B 《Protein science : a publication of the Protein Society》2003,12(7):1313-1322
The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest. 相似文献
20.
《Gene》1996,170(1):145-146
The Arg-encoding triplet (AGG) in the recognition sequence Ile-Glu-Gly-Arg for factor Xa can be used to generate a StuI restriction site (AGGCCT) which greatly facilitates the construction of DNA fragments encoding fusion proteins. Following proteolytic cleavage with factor Xa, a protein with the desired N terminus can be obtained 相似文献