Biomedical subjects

Ying-Lai Wang

Publications and source records attributed to Ying-Lai Wang.

11 recordsLinked to original sources

PubMed2002

The effect of N-terminal changes on arginyl-tRNA synthetase from Escherichia coli.

An Asn(2) deleted mutant of Escherichia coli arginyl-tRNA synthetase deleted Asn(2) and a chimera mutant, in which the N-terminal 23 amino acid residues of yeast arginyl-tRNA synthetase were appended to the N-terminus of Escherichia coli synthetase, were synthesized and studied. The expression of the deletion and chimera mutants in Escherichia coli formed inclusion bodies, presumably due to improper folding of the proteins. Relative to the native enzyme, the deletion mutant showed full amino acid activation activity and a 26% reduction in aminoacylation activity, while the chimera mutant lost 93% and 96% activities in aminoacid activation and aminoacylation, respectively, and did not aminoacylate yeast tRNA(Arg) at all. The mutant deleted Asn(2) and Ile(3) was able to be expressed in Escherichia coli but not stable to be purified. The emission maximum wavelength in the fluorescence spectra of the chimera mutants shifted to longer one and the corresponding intensities decreased, when compared with those of the native enzyme. The data show that the conformation of the mutants are different and the tryptophan residues in the mutants are more exposed than those in the native enzyme. An estimate of the secondary structure of the mutant enzymes from their far ultraviolet CD spectra showed that the chimera mutant contained less alpha-helix, more beta-sheet and slightly higher fraction of random coil, as compared with the native enzyme. The results indicate that an intact N-terminal domain of E.coli arginyl-tRNA synthetase is important to its activity and correct folding.

Amino Acid Sequence↗

PubMed1999

A Novel System for Hyper Expression and Rapid Purification of Arginyl-tRNA Synthetase from Escherichia coli.

The gene argS encoding arginyl-tRNA synthetase (ArgRS) in Escherichia coli has been cloned into the vector pMFT7-5 which allows overproduction of ArgRS. The specific activity of the synthetase in the crude extract of E.coli JM109(DE3) transformant containing the plasmid pMFT7-argS was approximately 2 500 times that of JM109(DE3) (the host strain without the vector). The overproduced synthetase can be purified to homogeneity with the specific activity of 36 000 units/mg under native condition within one day by a two-step purification system of DEAE-Sepharose CL-6B Fast Flow and Blue Sepharose CL-6B chromatographies with an overall yield of 69%. Moreover, this system makes it very easy to incorporate efficiently the expensive labeled amino acids into this enzyme. This novel system can conveniently provide a large amount of purified enzyme with a much higher specific activity than before which may be beneficial to NMR and crystallographic studies on this enzyme.

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PubMed1999

Gene Cloning, Overproduction and Purification of Escherichia coli tRNA(Arg)(2).

A synthetic gene encoding Escherichia coli tRNA(Arg)(2) was inserted in a plasmid under the control of an isopropyl-beta, D-thiogalactopyranoside (IPTG)-inducible promotor, pTrc99B. In E.coli MT102 transformed by the above plasmid containing the target gene. TRNA(Arg)(2) was overproduced up to 30 fold of that of the host. In the transformant the quantity contained tRNA(Arg) increased 10 times and was 70% of the total tRNA. The tRNA(Arg)(2) was purified to 88% homogeneity by passing through a DEAE-Sephacel column, and then was purified by benzyl-DEAE cellulose column chromatography to a purity of 99% with an arginylation activity of 1 600 pmole/A(260) unit. Eighteen milligrams of tRNA(Arg)(2) could be obtained from 40 mg total tRNA which was obtained from four liters of overnight culture, and the yield of the purification was 62%. The accurate kinetic constants of aminoacylation of tRNA(Arg)(2) catalyzed by arginyl-tRNA synthetase were comparable with that of tRNA(Arg) from Sigma.

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PubMed1998

Effect of Deletion of 245 and 252 Arginines in E. coli Arginyl-tRNA Synthetase on Its Structure and Function.

In order to study structure and function of E.coli arginyl-tRNA synthetase(ArgRS), deletion of R245 and 252 in this enzyme was carried out by site-directed mutagenesis of gene coding for this enzyme, respectively. The genes, argSdeltar245 and argSdeltar252-encoding ArgRS mutant, ArgRSdeltaR245 and ArgRSdeltaR252 were obtained and cloned into pUC18 or pTrc99B, respectively. The expression of argSdeltar245 and the properties of the mutant ArgRSdeltaR245 were studied. The gene encoding ArgRS, argS was expressed as a soluble protein; however, ArgRS deleted R245 formed inclusion body during the expression of argSdeltar245 in E. coli. After dissolution of the inclusion body by 8 mol/L urea or 7 mol/L guandidine chloride, followed by renaturation by the methods of gel filtration or dilution, the specific activity of the mutant enzyme, ArgRSdeltaR245 was about 40 unit/mg, that is only 0.3% activity of the native enzyme. By detection of ultraviolet absorbance and difference spectroscopy and titration of DTNB, the conformations of the native and mutant enzymes were compared. It was shown that the tertiary structure of ArgRSdeltaR245 was more relaxed than that of ArgRS, and the chromophoric groups in the mutant enzyme were more exposed than that of the native enzyme. It may be the main reason of forming inclusion body during the mutant gene expression and losing activity of enzyme.

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PubMed1998

Study on the Overexpression of the Gene Encoding Arginyl-tRNA Synthetase under Induction.

Arginyl-tRNA synthetase (ArgRS) from E. coli was overproduced from transformant containing the gene encoding this enzyme (argS) by 550 fold higher than that from host cell. By site-directed mutagenesis the cleavage site for NcoI restriction endonuclease was introduced into the start codon of argS. The mutant gene was recombinated with plasmid pTrc99B under IPTG control. In the transformant containing the recombinated plasmid, argS can overexpressed 2 000 times higher than that in host cell. Through one step DEAE-Sepharose column chromatography, ArgRS was purified to be one band by SDS-PAGE and its specific activity was 15 000 u/mg, similar to reported values. The mutant ArgRS2ND which had the second residue asparagine replaced by aspartic acid showed no change of activity, kinetic constants nor the thermal and denaturational stabilities.

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PubMed1998

Cloning and Expression of the pga Gene of Bacillus megaterium in E. coli.

The pga gene was amplified by PCR from Bacillus megaterium CA4098, cloned into pKK223-3 vector and expressed in E. coli HB101. The pga sequence was also determined. The deduced amino acids sequence was compared with those of PGAs from other bacteria and showed that they were similar, especially the active sites were strongly conserved.

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PubMed1997

The Overproduction of Leucyl-tRNA Synthetase in E. coli and Its Purification.

The 3.2 kb gene (leuS) encoding for leucyl-tRNA synthetase (LeuRS) has been cloned from E.coli K-12, and overexpressed 35 times more than that in the host strain TG1. In order to further increase the production of LeuRS, two types of leuS with different length of the 3' flanking region: leuS1 has an additional 130 bp over that of leuS2 were ligated into pKK-233-2 and pTrc-99B, respectively. In E. coli TG1 transformant harboring the recombination plasmid pTrc-99B with leuS2, the yield of LeuRS was 135 times that in TG1 strain. The purified enzyme that showed one band on SDS-PAGE was obtained after two steps of column chromatography. During the construction of plasmid, a substitute of G for C was introduced at position of base 4 in the coding region of leuS, so that Gln2 of LeuRS was changed to Glu. This enzyme was designated LeuRS2E. The kinetic constants of LeuRS2E showed that the substitution has no effect on the enzyme activity and could be used in the studies of LeuRS as the native enzyme.

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PubMed1997

The Construction and Functional Study of Protein Kinase Inhibitor Phage.

A DNA fragment, which encoded the heat-stable protein kinase inhibitor (PKI) (5-24) of camp-dependent protein kinase (cAPK), was synthesized and cloned into phage display vector fe-tet-DOG1. Thus, PKI(5-24) was displayed on the surface of phage fd, which was termed PKI phage (cAPK inhibitor phage), in a form fused with gene III protein (g3p). It was showed that PKI phage not only repressed cAPK effectively, but also bound with the immobilized recombinant His(6)-tag mouse cAPK-Calpha( His(6)-mCalpha) specifically. The bound PKI phages could be quantitatively eluted under acidic conditions. Model affinity screening demonstrated that PKI phages could be selectively enriched from the mixture of PKI phage and wild-type phages (1:10(8)) using affinity chromatography of immobilized His(6)-mCalpha. These results suggest that selecting protein kinase inhibitor by phage display technique is feasible.

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PubMed1996

The Spectroscopic Study of E. coli Arginyl-tRNA Synthetase (ArgRS) and its Mutants.

The conformation of native enzyme ArgRS and its mutants ArgRS306KA, ArgRS306KR and ArgRS381KA was studied by absorbance spectrum, Difference spectrum with solvent perturbation, fluorescence spectrum and CD spectrum. The results showed that the chromophores of ArgRS306KR and ArgRS306KA had shifted to different micro environments in comparison to the native enzyme. Compared to the native enzyme, ArgRS306KA had and even bigger conformational change than ArgRS306KR, while the conformation difference between ArgRS381KA and ArgRS was not big enough to be perceivable in the spectrum. The analysis of CD spectrum showed the less the percentage of beta-turn in the ArgRS mutants, the lower the activity of the mutants. The conclusion can be drawn that the positive charge of Lys306 is very important to maintain the conformation of ArgRS, the change of conformation might be mainly responsible for the loss of enzyme of the mutants, while the substitution of Lys381 seems to cause no perceivable change of the enzyme conformation.

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PubMed1996

The Construction of T7 Promoter-based His(6)-tagging Vectors and the Single-step Purification of the Expression products.

T7 promoter-based fusion expression vectors have been constructed that directed the synthesis of heterologous proteins in E. coli as fusions with a stretch of six consecutive histidine residues His(6) at N Terminus. The vectors were also featured with strong T7 promoter, terminator, translational start, multiple cloning sites with seven unique restriction sites in all three reading frames and the f1 phage origin which allows the packaging of single-stranded plasmid, mutagenesis and DNA sequencing without subcloning steps. In most cases, expressed fusion proteins are soluble. The His(6) tag allows the fusion proteins purified in one step by immobilized metal (Ni(2+)) chelation affinity chromatography in the denatured or native state. As an example of the general utility of these expression vectors, the His(6)-fused catalytic subunits of mouse cAMP-dependent protein kinase were expressed with high activity by using these vectors and could be purified to homogeneity in one step.

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PubMed1996

A T7 Promoter-based Versatile Expression Vector System.

A family of T7 promoter-based versatile expression plamides for E. coli Were constructed. These vectors were featured strong T7 promoter, translational start, stop elements, a multiple cloning site with eight unique restriction sites in all three reading frames and f1 phage origin which allows packaging of single stranded plasmid, mutagensis and gene sequencing without subcloning steps. With these vectors recombinant proteins can be expressed in mature or short fusion forms. Many heterolegous genes have been highly expressed using these vectors, most of them were expressed in soluble and active forms.

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