Characterization of the starch branching enzymes in Arabidopsis thaliana

The roles of three starch branching enzyme isoforms, BE1, BE2 and BE3, in Arabidopsis leaf starch metabolism were investigated by AtBE mutant analysis. We also determined the expression patterns of BEs in Arabidopsis by GUS staining and microarray experiments. The possible correlation of BE gene expression with other genes in the Arabidopsis genome was analyzed to identify putative genes that are functionally correlated with BEs. Motifs in the promoters of the Arabidopsis BE genes were identified to better understand their expression differences. BE2 and BE3 expression levels during a diurnal cycle were evaluated with isoform specific antibodies. Meantime, the in vitro biochemical activities of purified recombinant BE2 and BE3 proteins were investigated by three different assays to better understand their specific functions in starch biosynthesis. Analyses of the structures of the final products from the in vitro BE-amylose reactions were done by FACE with two different forms of amylose

The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications

The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy

Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

<p>Abstract</p> <p>Background</p> <p>Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step.</p> <p>Results</p> <p>A recombinant human collagen type I α-1 (rCIα1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1.</p> <p>Conclusions</p> <p>This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.</p

Characterization of the starch branching enzymes in Arabidopsis thaliana

The roles of three starch branching enzyme isoforms, BE1, BE2 and BE3, in Arabidopsis leaf starch metabolism were investigated by AtBE mutant analysis. We also determined the expression patterns of BEs in Arabidopsis by GUS staining and microarray experiments. The possible correlation of BE gene expression with other genes in the Arabidopsis genome was analyzed to identify putative genes that are functionally correlated with BEs. Motifs in the promoters of the Arabidopsis BE genes were identified to better understand their expression differences. BE2 and BE3 expression levels during a diurnal cycle were evaluated with isoform specific antibodies. Meantime, the in vitro biochemical activities of purified recombinant BE2 and BE3 proteins were investigated by three different assays to better understand their specific functions in starch biosynthesis. Analyses of the structures of the final products from the in vitro BE-amylose reactions were done by FACE with two different forms of amylose.</p

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field

Genome-Wide Screening of Oxidizing Agent Resistance Genes in <i>Escherichia coli</i>

The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins