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Zhiwen Zhang

Publications and source records attributed to Zhiwen Zhang.

10 recordsLinked to original sources
PubMed2004

RETRACTED: A new strategy for the synthesis of glycoproteins.

Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing beta-N-acetylglucosamine (GlcNAc)-serine at a defined position can be expressed in Escherichia coli in good yield and with high fidelity. The beta-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.

Acetylation↗
PubMed2003

Site-specific incorporation of a redox-active amino acid into proteins.

The redox-active amino acid 3,4-dihydroxy-l-phenylalanine (DHP), which can undergo two-electron oxidation to a quinone, has been incorporated selectively and efficiently into proteins in Escherichia coli in response to a TAG codon. We have demonstrated that DHP can be oxidized electrochemically within the protein. The ability to incorporate a redox-active amino acid site specifically into proteins should facilitate the study of electron transfer in proteins, as well as enable the engineering of redox proteins with novel properties.

Amino Acyl-tRNA Synthetases↗
PubMed2003

An expanded eukaryotic genetic code.

We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.

Amino Acids↗
PubMed2003

A new strategy for the site-specific modification of proteins in vivo.

We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed2003

Targeting gene expression of the mouse uroplakin II promoter to human bladder cells.

Differential expression of the desired gene product in the target tissue is central to the concept of gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic genes. To investigate the feasibility of tissue-specific gene therapy for bladder cancer using the mouse uroplakin II (UPII) promoter and its transcriptional control, the efficacy of this promoter as well as fragments in regulating gene expression were qualitatively and quantitatively analyzed in bladder and non-bladder tissue cell lines using DNA transfection. Our results demonstrate that the mouse UPII promoter actively drives gene expression in BIU-87, a bladder cancer cell line. Little promoter activity was detected in the non-bladder tissue cell lines. Furthermore, deleting the 5' end 1.5 kb of the UPII promoter by PCR, the activity was significantly decreased but was bladder-specific. However, deleting the 3' end 143-bp of the UPII promoter, the activity was hardly detected in any tissue cell lines. The activity of the 3' end 143-bp of the UPII promoter was detected in both bladder cancer and stomach cancer cell lines. These data demonstrate that the mouse UPII promoter has a high activity in human bladder cells and a low basal activity in human non-bladder cells. This suggests that targeting the gene expression of the mouse UPII promoter could be used to treat human bladder cancer. The enhancer was contained in the region of the 1.5 kb of the 5' end of the mouse UPII promoter. The core promoter was located in the region of the 143 bp of the 3' end.

Animals↗
PubMed2003

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban.

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.

Buffers↗
PubMed2003

Evaluation of MCAO stroke models in normotensive rats: standardized neocortical infarction by the 3VO technique.

The temporary three-vessel occlusion (3VO) technique with a surgical approach for middle cerebral artery (MCA) produces consistent cerebral infarction in the neocortex in normotensive rats. The intraluminal thread-occlusion technique with an endovascular approach targeting the MCA occlusion (MCAO) is more widely used since it does not require complicated intracranial procedures. The aim of this study was to review the methods/models for MCAO stroke in normotensive rats and to evaluate a 3VO stroke model that provides consistent degrees and variance of cortical stroke injury for additional discussion. First, we analyzed a model with modified temporary 3VO technique requiring less complicated procedures than the temporary 3VO model, i.e., temporary occlusion of the bilateral common carotid arteries (CCAs) superimposed on a permanent occlusion of the MCA, in Sprague-Dawley rats or C57BL/6J mice. In the microvascular tissue (cerebral) perfusion study, significant reductions in regional cerebral perfusion during the 3VO accompanied a rapid return to baseline after release of the CCAs, showing that the technique induces temporary focal ischemia. The average sizes and variances of the neocortical infarction in this model, together with those in the other normotensive rat models caused by the 3VO technique in the literature, indicated a standard size and variance of infarcted lesion in the control groups relative to the specific ischemic period. However, stroke injuries in the neocortex induced by the thread occlusion technique showed greater variability with less consistent lesion sizes. Inclusion/exclusion criteria to avoid inappropriate cases with too mild (no/faint infarction) or too great (huge/fatal infarction) severity in the ischemic injury may differ between laboratories in the thread occlusion model.

Animals↗
PubMed2002

Addition of the keto functional group to the genetic code of Escherichia coli.

Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Amino Acid Substitution↗
PubMed2002

Renal cell carcinoma related novel gene, GYLZ-RCC18: cloning and functional studies.

OBJECTIVE: To clone the full length of renal cell carcinoma (RCC) related novel gene GYLZ-RCC18 and study its function. METHODS: SMART RACE technology was used to clone the full length of GYLZ-RCC18. RT-PCR was used to detect its expression in renal cell carcinoma tissue at different stages and grades. We transfected the antisense oligonucleotide of GYLZ-RCC18 to renal cell carcinoma cell line, GRC-1, and analyzed proliferation activity, growth rate, apoptosis, and mortality changes. RESULTS: The full length of GYLZ-RCC18 (GenBank accession number: BE825133) cDNA was about 3.5 kb. GYLZ-RCC18 had a higher expression in higher grades and stages of renal cell carcinoma than in lower ones. The expression of GYLZ-RCC18 in renal cell carcinoma was much higher than in normal kidney. After the transfection of GYLZ-RCC18 antisense oligonucleotide, the mortality of GRC-1 increased significantly, while proliferative activity and growth rate were substantially inhibited at the same time. The antisense oligonucleotide induced apoptosis of GRC-1 through the entire observation time. CONCLUSION: GYLZ-RCC18 is an important novel gene related to renal cell carcinoma. Overexpression of this gene results in higher growth and proliferative activity and has an antiapoptosis effect on renal cell carcinoma cells. Transfection of the antisense oligonucleotide may inhibit the generation and development of renal cell carcinoma.

Apoptosis↗