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Histidine Tag (histidine + tag)
Selected AbstractsPotential for Using Histidine Tags in Purification of Proteins at Large ScaleCHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005V. Gaberc-Porekar Abstract Attachment of oligo-histidine tag (His-tag) to the protein N- or C-terminus is a good example of early and successful protein engineering to design a unique and generalized purification scheme for virtually any protein. Thus relatively strong and specific binding of His-tagged protein is achieved on an Immobilized Metal-Ion Affinity Chromatography (IMAC) matrix. Most popular hexa-histidine tag and recently also deca-histidine tag are used in combination with three chelating molecules: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), and carboxymethyl aspartic acid (CM-Asp), covalently attached to the chromatographic matrix. The following combinations with divalent metal ions are preferentially used: (Cu, Zn, Ni, Co)-IDA, Ni-NTA, and Co-CM-Asp. At large scale, regarding cost and product purity, a decisive step is precise and efficient cleavage of His-tag by the cleavage enzyme. Two-step IMAC followed by a polishing step appears to be a minimum but still realistic as an approach to generic technology also for more demanding products. Possible drawbacks in using His-tags and IMAC, such as leaching of metal ions, inefficient cleavage, and batch-to-batch reproducibility must be carefully evaluated before transferred to large scale. Although a great majority of reports refer to small laboratory scale isolations for research purposes it appears there is much higher potential for more extensive use of His-tags and IMAC at large scale than currently documented. [source] The lipase/acyltransferase from Candida parapsilosisFEBS JOURNAL, Issue 6 2002Molecular cloning, characterization of purified recombinant enzymes Candida parapsilosis has been previously shown to produce a lipase (i.e. able to catalyze efficiently the hydrolysis of insoluble lipid esters such as triacylglycerols) that preferentially catalyses transfer reactions such as alcoholysis in the presence of suitable nucleophiles other than water, even in aqueous media with high (> 0.9) water thermodynamic activity. The present work describes the cloning and the overexpression of the gene coding for this enzyme. Two ORFs (CpLIP1 and CpLIP2) were isolated. The deduced 465-amino-acid protein sequences contained the consensus motif (G-X-S-X-G) which is conserved among lipolytic enzymes. Only one of the two deduced proteins (CpLIP2) contained peptide sequences obtained from the purified lipase/acyltransferase. Homology investigations showed that CpLIP2 has similarities principally with 11 lipases produced by C. albicans (42,61%) and the lipase A from Candida antarctica (31%) but not with the other lipases sequenced so far. Both CpLIP1 and CpLIP2 were expressed in Saccharomyces cerevisiae, but only CpLIP2 coded for an active protein. The substrate specificity and the catalytic behavior of purified recombinant CpLIP2, with or without a C-terminal histidine tag, were not changed compared to those of the native lipase. [source] Purification and characterization of the heterologously expressed trehalose/maltose ABC transporter complex of the hyperthermophilic archaeon Thermococcus litoralisFEBS JOURNAL, Issue 14 2001Gerhard Greller We report the purification of the maltose/trehalose transporter complex MalFGK of the hyperthermophilic archaeon Thermococcus litoralis. The complex was expressed in Escherichia coli, solubilized in dodecyl maltoside and purified with the aid of a histidine tag on one of the membrane proteins. One hundred grams of cells yielded 3 mg of pure complex. The final product showed ATPase activity at 70 °C and was soluble at low detergent concentration. ATPase activity was not due to dissociation of the MalK subunit from the integral membrane proteins MalF and MalG but could not be further stimulated by trehalose/maltose binding protein (TMBP), be it the native protein as isolated from T. litoralis or the soluble engineered protein. The purified native TMBP was identified as a glycoprotein. [source] Overexpression and Characterization of the Rhodobacter sphaeroides PufX Membrane Protein in Escherichia coli,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2007Shiho Onodera Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had ,-helix contents of 29% in organic solvents and 22,26% in 0.8,2.0% (w/v) n -octyl ,- d -glucopyranoside solutions, suggesting that the PufX contains a substantial ,-helical region composed of 18,22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 ,- and ,-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system. [source] Pharaonis Phoborhodopsin Binds to its Cognate Truncated Transducer Even in the Presence of a Detergent with a 1:1 Stoichiometry,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2001Yuki Sudo ABSTRACT Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis.ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6× histidine tag) form a complex even in the presence of 0.1% of n -dodecyl-,- d -maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 ± 1.8 ,M) and the number of binding sites (1.2 ± 0.08). [source] Structures of the B1 domain of protein L from Peptostreptococcus magnus with a tyrosine to tryptophan substitutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2001Jason W. O'Neill The three-dimensional structure of a tryptophan-containing variant of the IgG-binding B1 domain of protein L has been solved in two crystal forms to 1.7 and 1.8,Å resolution. In one of the crystal forms, the entire N-terminal histidine-tag region was immobilized through the coordination of zinc ions and its structural conformation along with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overall structure of the rest of the protein. Structural comparisons of the tryptophan-containing variant with an NMR-derived wild-type structure, which contains a tyrosine at position 47, reveals a common fold, although the overall backbone root-mean-square difference is 1.5,Å. The Y47W substitution only caused local rearrangement of several side chains, the most prominent of which is the rotation of the Tyr34 side chain, resulting in a 6,Å displacement of its hydroxyl group. A small methyl-sized cavity bounded by ,-strands 1, 2 and 4 and the ,-helix was found in the structures of the Y47W-substituted protein L B1 domain. This cavity may be created as the result of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resolution structures of the tryptophan-containing variant provide a reference frame for the analysis of thermodynamic and kinetic data derived from a series of mutational studies of the protein L B1 domain. [source] Crystallization and crystal-packing studies of Chlorella virus deoxyuridine triphosphataseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Kohei Homma The 141-amino-acid deoxyuridine triphosphatase (dUTPase) from the algal Chlorella virus IL-3A and its Glu81Ser/Thr84Arg-mutant derivative Mu-22 were crystallized using the hanging-drop vapor-diffusion method at 298,K with polyethylene glycol as the precipitant. An apo IL-3A dUTPase with an amino-terminal T7 epitope tag and a carboxy-terminal histidine tag yielded cubic P213 crystals with unit-cell parameter a = 106.65,Å. In the presence of dUDP, the enzyme produced thin stacked orthorhombic P222 crystals with unit-cell parameters a = 81.0, b = 96.2, c = 132.8,Å. T7-histidine-tagged Mu-22 dUTPase formed thin stacked rectangular crystals. Amino-terminal histidine-tagged dUTPases did not crystallize but formed aggregates. Glycyl-seryl-tagged dUTPases yielded cubic P213 IL-3A crystals with unit-cell parameter a = 105.68,Å and hexagonal P63 Mu-22 crystals with unit-cell parameters a = 132.07, c = 53.45,Å, , = 120°. Owing to the Thr84Arg mutation, Mu-22 dUTPase had different monomer-to-monomer interactions to those of IL-3A dUTPase. [source] Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005N. Abdullah Abstract Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His6 by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His6 to MBP was 16 h, as judged by SDS,PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc. [source] His-tagged protein purification by metal-chelate affinity extraction with nickel-chelate reverse micellesBIOTECHNOLOGY PROGRESS, Issue 4 2010Xiao-Yan Dong Abstract Di(2-ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X-45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R2. The Ni(II)-chelate reverse micelles were characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R2 was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni2+ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two-stage purification by the metal-chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009J.J. Leisner Abstract Aims:, To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results:, The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N,-diacetyl-,- d -chitobioside with Km values of 1·6 and 2·1 mmol l,1, respectively, and kcat values of 21·6 and 6·5 s,1. The enzymes also showed activity towards 4-nitrophenyl ,- d - N, N,, N,-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N- acetyl-,- d -glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl ,- d -cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. Conclusion:, The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. Significance and Impact of the Study:, This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants. [source] | |||||||||||||