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Chromatographic Matrices (chromatographic + matrix)
Selected AbstractsIdentification of Trichoderma strains by image analysis of HPLC chromatogramsFEMS MICROBIOLOGY LETTERS, Issue 2 2001Ulf Thrane Abstract Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain , except one , was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification. [source] Preparation of novel acrylamide-based thermoresponsive polymer analogues and their application as thermoresponsive chromatographic matricesJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 16 2008Yoshikatsu Akiyama Abstract New thermoresponsive polymers based on poly(N -(N, -alkylcarbamido)propyl methacrylamide) analogues were designed with increased hydrophobic content to facilitate temperature-dependent chromatographic separations of peptides and proteins from aqueous mobile phases. These polymer solution exhibited a lower critical solution temperature (LCST) when the alkyl group is methyl, ethyl, isopropyl, propyl, butyl, and isobutyl. However, larger alkyl groups such as hexyl and phenyl were not soluble in aqueous solutions at any temperature. Phase transition temperatures were lower for larger alkyl groups and increased with decreasing polymer molecular weight and concentration in solution. LCST dependence on polymer molecular weight and concentration is more significant compared with well-studied poly(N -isopropylacrylamide) (PIPAAm). Partition coefficient (log P) values for N -(N, -butylcarbamide)propylmethacrylamide and N -(N, -isobutylcarbamide)propyl methacrylamide (iBuCPMA) monomers are larger than that for IPAAm monomer, suggesting higher hydrophobicity than IPAAm. Chromatographic evaluation of poly(N -(N, -isobutylcarbamide)propyl methacrylamide) (PiBuCPMA) grafted silica particles in aqueous separations revealed larger k, values for peptides, insulin, insulin chain B, and angiotensin I than PIPAAm-grafted silica beads. In particular, k, values for insulin obtained from PiBuCPMA-grafted silica separations were much larger than those from PIPAAm-grafted surface separations, indicating that PiBuCPMA should be more hydrophobic than PIPAAm. These results support the introduction of alkylcarbamido groups to efficiently increase thermoresponsive polymer hydrophobicity of poly(N -alkylacrylamides) and poly(N -alkylmethacrylamides). Consequently, poly(N -(N, -alkylcarbamido)propyl methacrylamide) analogues such as PiBuCPMA and poly(N -(N, -alkylcarbamido)alkylmehacrylamide) are new thermoresponsive polymers with appropriate hydrophobic partitioning properties for protein and peptide separations in aqueous media, depending on selection of their alkyl groups. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 5471,5482, 2008 [source] Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] Potential 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] | ||||||||||