A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.
"Large-scale production of AtXTH31 and subsequent affinity purification were performed according to the methods described by Baumann et al. (2007). Protein electrospray mass spectrometry was performed as described by Sundqvist et al. (2007). "
[Show abstract][Hide abstract] ABSTRACT: The molecular basis of primary wall extension endures as one of the central enigmas in plant cell morphogenesis. Leading cell wall models suggest that xyloglucan endo-transglycosylase (XET) activity is the primary catalyst (together with expansins) of controlled cell wall loosening through the transient cleavage and re-ligation of xyloglucan-cellulose cross-links. The genome of Arabidopsis thaliana contains 33 phylogenetically diverse XYLOGLUCAN ENDO-TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products, two of which were predicted to be predominant xyloglucan endo-hydrolases (XEH), due to clustering into Group III-A. Enzyme kinetic analysis of recombinant AtXTH31 confirmed this prediction, and indicated that this enzyme had similar catalytic properties to the Tropaeolum majus (nasturtium) xyloglucanase 1 (TmNXG1) responsible for storage xyloglucan hydrolysis during germination. Global analysis of Genevestigator data indicated that AtXTH31 and the paralogous AtXTH32 were abundantly expressed in expanding tissues. Microscopy analysis, utilizing the resorufin beta-glycoside of the xyloglucan oligosaccharide XXXG as an in situ probe, indicated significant XEH activity in specific regions of both roots and hypocotyls, in good correlation with transcriptomic data. Moreover, this hydrolytic activity was essentially completely eliminated in AtXTH31/AtXTH32 double-knock-out lines. However, single- and double-knock-out lines, as well as individual over-expressing lines, of AtXTH31 and AtXTH32 did not demonstrate significant growth nor developmental phenotypes. These results suggest that although xyloglucan polysaccharide hydrolysis occurs in parallel with primary wall expansion, morphological effects are subtle or may be compensated by other mechanisms. We hypothesize that there is likely to be an interplay between these xyloglucan endo-hydrolases and recently discovered apoplastic exo-glycosidases in the hydrolytic modification of matrix xyloglucans.
"Two MS methods can be used to determine the intact mass of both microbial and plant proteins: electrospray MS, often implemented on a quadrupole-time-of-flight (Q- TOF) type mass spectrometer, and Matrix Assisted Laser Desorbtion Ionisation (MALDI) MS on a MALDI-TOF instrument (Sundqvist et al. 2007). For MS analysis of microbial proteins, Q-TOF analysis is preferred because it achieves higher mass accuracy than MALDI-TOF analysis (Sundqvist et al. 2007). Q-TOF machines are able to distinguish between proteins with single amino acid substitutions, or other low molecular weight modifications, such as methionine oxidation. "
[Show abstract][Hide abstract] ABSTRACT: Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.
Transgenic Research 10/2012; 22(2). DOI:10.1007/s11248-012-9658-3 · 2.32 Impact Factor
"Large-scale production of AtXTH13 and subsequent affinity purification was performed according to the methods described by Baumann et al. (2007). Protein electrospray mass spectrometry was performed as described by Sundqvist et al. (2007). XET dot-blot activity assay A dot-blot assay (Fry, 1997) was used to test the xyloglucan endotransglucosylase (XET) activity of the recombinant proteins. "
[Show abstract][Hide abstract] ABSTRACT: Xyloglucan endotransglucosylase/hydrolases (XTHs) are cell wall enzymes that are able to graft xyloglucan chains to oligosaccharides or to other available xyloglucan chains and/or to hydrolyse xyloglucan chains. As they are involved in the modification of the load-bearing cell-wall components, they are believed to be very important in the regulation of growth and development. Given the large number (33) of XTH genes in Arabidopsis and the overlapping expression patterns, specific enzymic properties may be expected. Five predominantly root-expressed Arabidopsis thaliana XTHs belonging to subgroup I/II were analysed here. These represent two sets of closely related genes: AtXTH12 and 13 on the one hand (trichoblast-enriched) and AtXTH17, 18, and 19 on the other (expressed in nearly all cell types in the root). They were all recombinantly produced in the yeast Pichia pastoris and partially purified by ammonium sulphate precipitation before they were subsequently all subjected to a series of identical in vitro tests. The kinetic properties of purified AtXTH13 were investigated in greater detail to rule out interference with the assays by contaminating yeast proteins. All five proteins were found to exhibit only the endotransglucosylase (XET; EC 184.108.40.206) activity towards xyloglucan and non-detectable endohydrolytic (XEH; EC 220.127.116.11) activity. Their endotransglucosylase activity was preferentially directed towards xyloglucan and, in some cases, water-soluble cellulose acetate, rather than to mixed-linkage β-glucan. Isoforms differed in optimum pH (5.0-7.5), in temperature dependence and in acceptor substrate preferences.
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