共查询到20条相似文献,搜索用时 24 毫秒
1.
Srivastava R Liu JX Howell SH 《The Plant journal : for cell and molecular biology》2008,56(2):219-227
Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture. 相似文献
2.
The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins. 相似文献
3.
Ren X Zhao L Sivashanmugam A Miao Y Korando L Yang Z Reardon CA Getz GS Brouillette CG Jerome WG Wang J 《Biochemistry》2005,44(45):14907-14919
Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination. 相似文献
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6.
Engineering subtilisin BPN' for site-specific proteolysis 总被引:6,自引:0,他引:6
A combination of protein engineering and substrate optimization was used to create variants of the serine protease, subtilisin BPN', which efficiently and specifically cleave a designed target sequence in a fusion protein. The broad substrate specificity of wild-type subtilisin BPN' is greatly restricted by substitution of the catalytic histidine-containing of the catalytic histidine 64 with alanine (H64A) so that certain histidine-containing substrates are preferentially hydrolysed (Carter, P., Wells, J.A. Science 237:394-399, 1987). The catalytic efficiency, (kcat/Km), of this H64A variant was increased almost 20-fold by judicious choice of substrate and by installing three additional mutations which increase the activity of wild-type subtilisin. The most favorable substrate sequence identified was introduced as a linker in a fusion protein between a synthetic IgG binding domain of Staphylococcus aureus protein A and Escherichia coli alkaline phosphatase. The fusion protein (affinity purified on an IgG column) was cleaved by the prototype H64A enzyme and its improved variant, efficiently and exclusively at the target site, to liberate an alkaline phosphatase product of the expected size and N-terminal sequence. Several features of H64A variants of subtilisin make them attractive for site-specific proteolysis of fusion proteins: they have exquisite substrate specificity on the N-terminal side of the cleavage site and yet are broadly specific on the C-terminal side; they can be produced in large quantities and remain highly active even in the presence of detergents, reductants (modest concentrations), protease inhibitors, at high temperatures, or when specifically immobilized on a solid support. 相似文献
7.
Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions. 相似文献
8.
Felix Jakob Ronny Martinez John Mandawe Hendrik Hellmuth Petra Siegert Karl-Heinz Maurer Ulrich Schwaneberg 《Applied microbiology and biotechnology》2013,97(15):6793-6802
In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. “Deamidation” in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5–10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH?8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance. 相似文献
9.
While protein purification has long been dominated by standard chromatography, the relatively high cost and complex scale‐up have promoted the development of alternative non‐chromatographic separation methods. Here we developed a new non‐chromatographic affinity method for the purification of proteins expressed in Escherichia coli. The approach is to genetically fuse the target proteins with an affinity tag. Direct purification and recovery can be achieved using a thermo‐responsive elastin‐like protein (ELP) scaffold containing the capturing domain. Naturally occurring cohesin–dockerin pairs, which are high‐affinity protein complex responsible for the formation of cellulosome in anaerobic bacteria, were used as the model. By exploiting the highly specific interaction between the dockerin and cohesin domain from Clostridium thermocellum and the reversible aggregation property of ELP, highly purified and active dockerin‐tagged proteins, such as the endoglucanase CelA, chloramphenicol acetyl transferase (CAT), and enhanced green fluorescence protein (EGFP), were recovered directly from crude cell extracts in a single thermal precipitation step with yields achieving over 90%. Incorporation of a self‐cleaving intein domain enabled rapid removal of the affinity tag from the target proteins, which was subsequently removed by another cycle of thermal precipitation. This method offers great flexibility as a wide range of affinity tags and ligands can be used. Biotechnol. Bioeng. 2012; 109: 2829–2835. © 2012 Wiley Periodicals, Inc. 相似文献
10.
Xiang Gao Ming‐Yang Zhou Peng Wang Hui‐Lin Zhao Bin‐Bin Xie Xi‐Ying Zhang Xiu‐Lan Chen Bai‐Cheng Zhou Yu‐Zhong Zhang 《Molecular microbiology》2013,90(5):997-1010
A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP‐01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP‐01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate‐binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP‐01 displayed a non‐strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1’ site in collagen. His211 is a key residue for the P1‐basic‐residue preference of MCP‐01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms. 相似文献
11.
An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence
for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized
for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence
but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both
fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove
protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude
cell extract. 相似文献
12.
Concentrating alkaline serine protease,subtilisin, using a temperature-sensitive hydrogel 总被引:2,自引:0,他引:2
Summary The alkaline serine protease, subtilisin, produced by Bacillus licheniformis was concentrated using hydrogel ultrafiltration. Separation efficiency at 15° and 20°C was 84 % but decreased above 25°C. 相似文献
13.
Beyond the rewards of plant genome analysis and gene identification, characterisation of protein activities, post-translational modifications and protein complex composition remains a challenge for plant biologists. Ideally, methods should allow rapid isolation of proteins from plant material achieving a high degree of purity. We tested three purification strategies based on the eight-amino acid StrepII, six-amino acid His6 and 181-amino acid Tandem Affinity Purification (TAP) affinity tags for enrichment of a membrane-anchored protein kinase, NtCDPK2, and a soluble protein, AtSGT1b, from leaf extracts. Transiently expressed StrepII-taggedNtCDPK2 was purified from Nicotiana benthamiana to almost complete homogeneity in less than 60min and was directly suitable for enzymatic or mass-spectrometric analyses, allowing the identification of in planta phosphorylation sites. In contrast, purification of NtCDPK2 via His6 tag yielded partially oxidised protein of low purity. AtSGT1b could be isolated after transient expression from N. benthamiana or from transgenic Arabidopsis thaliana as either TAP-tagged or StrepII-tagged protein. While StrepII-tag purification achieved similar yield and high purity as the TAP-tag strategy, it was considerably easier and faster. Using either tagging strategy, a protein was co-purified with AtSGT1b from N. benthaniana and A. thalianaleaf extracts, suggesting that both the StrepII and TAP tags are suitable for purification of protein complexes from plant material. We propose that the StrepII epitope, in particular, may serve as a generally utilizable tag to further our understanding of protein functions, post-translational modifications and interaction dynamics in plants. 相似文献
14.
A general, convenient, one-step purification procedure for chemically synthesized proteins present in low yields using on-resin biotinylation is reported. The protein, terminally deprotected and neutralized on-resin, is stirred in dimethylformamide and then biotinylated with N-hydroxysuccinimidobiotin (2 mg/mg protein on-resin) for 24 h at 45 degrees C. Following low/high hydrogen fluoride cleavage (J. P. Tam, W. F. Heath, and R. B. Merrifield (1983) J. Amer. Chem. Soc. 105, 6442-6455) the crude cleavage product was applied to an avidin agarose column. The column was washed with phosphate-buffered saline until all unbound materials had been eluted off. Then the biotinylated protein was eluted with 0.1 M glycine HCl, pH 2.0. A pilot experiment with two unrelated peptides on-resin established the experimental conditions for biotinylation. We then demonstrated that the chemically synthesized 153 residue [Asp205]-interleukin-1 beta (117-269), present in less than 1% yield in the crude HF cleavage mixture, could be purified to homogeneity in one step. In addition 70 and 114 residue synthetic fragments, (200-269) and (156-269), were also purified in this manner. Biotinylation on-resin appears to be an attractive method of purifying low yield chemically synthesized proteins and for preparing proteins with biotinyl moieties at specific locations such as the amino terminus. 相似文献
15.
S. Sonezaki A. Kondo T. Oba Y. Ishii Y. Kato H. Nakayama 《Applied microbiology and biotechnology》1994,42(2-3):313-318
Lon protease, which plays a major role in degradation of abnormal proteins inEscherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector. The MBP-Lon fusion protein was expressed in a soluble form inE. coli and purified to homogeneity by amylose resin in a single step. Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration. In this simple method, Lon protease was purified to homogeneity. Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-l-phenylalanyl-l-leucyl-phenylalanyl--d-methoxynaphthylamide) and protein (-casein) in the presence of ATP. Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease. MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes. 相似文献
16.
《Process Biochemistry》2010,45(2):292-296
Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation. 相似文献
17.
Polyprotein processing protease of African swine fever virus: purification and biochemical characterization
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The purified recombinant African swine fever virus polyprotein processing protease cleaves the two GG-X sites in polyprotein pp62 with the same efficiency. Cleavage at the site that is first recognized in vivo is not a requisite for cleavage at the second site, suggesting the existence of mechanisms that control the ordered processing of the polyprotein during infection. 相似文献
18.
Karpol A Kantorovich L Demishtein A Barak Y Morag E Lamed R Bayer EA 《Journal of molecular recognition : JMR》2009,22(2):91-98
Efficient degradation of cellulose by the anaerobic thermophilic bacterium, Clostridium thermocellum, is carried out by the multi-enzyme cellulosome complex. The enzymes on the complex are attached in a calcium-dependent manner via their dockerin (Doc) module to a cohesin (Coh) module of the cellulosomal scaffoldin subunit. In this study, we have optimized the Coh-Doc interaction for the purpose of protein affinity purification. A C. thermocellum Coh module was thus fused to a carbohydrate-binding module, and the resultant fusion protein was applied directly onto beaded cellulose, thereby serving as a non-covalent "activation" procedure. A complementary Doc module was then fused to a model protein target: xylanase T-6 from Geobacillus stearothermophilus. However, the binding to the immobilized Coh was only partially reversible upon treatment with EDTA, and only negligible amounts of the target protein were eluted from the affinity column. In order to improve protein elution, a series of truncated Docs were designed in which the calcium-coordinating function was impaired without appreciably affecting high-affinity binding to Coh. A shortened Doc of only 48 residues was sufficient to function as an effective affinity tag, and highly purified target protein was achieved directly from crude cell extracts in a single step with near-quantitative recovery of the target protein. Effective EDTA-mediated elution of the sequestered protein from the column was the key step of the procedure. The affinity column was reusable and maintained very high levels of capacity upon repeated rounds of loading and elution. Reusable Coh-Doc affinity columns thus provide an efficient and attractive approach for purifying proteins in high yield by modifying the calcium-binding loop of the Doc module. 相似文献
19.
Fuentes M Batalla P Grazu V Pessela BC Mateo C Montes T Hermoso JA Guisan JM Fernandez-Lafuente R 《Biomacromolecules》2007,8(2):703-707
A support having similar amounts of carboxymethyl and amino groups has been prepared and evaluated as an ion exchanger. It has been found that this support was able to adsorb a high amount of protein from a crude extract of proteins (approximately 55%) at pH 5. Moreover, it was able to adsorb approximately 60% of the protein that did not become adsorbed on supports bearing just one kind of ionic groups. The use of divalent cations reinforced the adsorption of proteins on these supports. These results suggest that the adsorption of proteins on supports bearing almost neutral charge is not driven by the existence of opposite charges between the adsorbent and the biomacromolecule but just by the possibility of forming a high number of enzyme-support ionic bonds. This support has been used to purify the enzyme penicillin G acylase (PGA) from Escherichia coli. PGA was not significantly adsorbed at any pH value on either amino- or carboxyl-activated supports, while it can be fully adsorbed at pH 5 on this new carboxyl-amino matrix. Thus, we have been able to almost fully purify PGA from crude extracts with a very high yield by using these new supports. 相似文献
20.
With the availability of new and improved DNA vectors for producing large amounts of proteins by recombinant DNA technology, there is a growing need for efficient recovery of the desired protein, often from liters of culture medium. In this report, we describe a relatively simple modification of recombinant cDNA, that, in combination with immobilized metal affinity chromatography, is a rapid, inexpensive and highly effective method for enriching or purifying a desired protein. 相似文献