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Bo-Liang Li

Publications and source records attributed to Bo-Liang Li.

30 records · Page 2Linked to original sources
PubMed1998

C-terminal His-tag Fusion Expression and Purification of Truncated cAMP-dependent Protein Kinase.

The truncated mCalpha lacking 3'-coding sequence of 96 base paires was fused with a His-tag (mCalpha4H) and a C-terminal fusion expression plasmid pZP mCalpha4H was constructed. With the induction of IPTG, the Expressed mCalpha4H was up to about 20% of the total bacterial proteins in E. coli BL21 (DE3). Using immobilized metal (Ni(2+)) chelation affinity chromatography, the target protein mCalpha4H was purified from crude lysates and inclusion bodies respectively. The results of in vitro and in vivo myristoylation assay showed that the purifed mCalpha4H is a substrate of NMT as the mCalpha.

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PubMed1998

Selection of Myristoyltransferase Inhibitor Phages from Phage Display Random Peptide Library.

Through screening library with either immobilized or coated enzymes, we selected disease-related myristoyltransferase inhibitor phages from phage display random peptide library. After high-affinity bound phages were obtained, they were subjected to in vitro NMT inhibition assay to identify inhibitor phages. The results of DNA sequencing, peptide sequence deducing and sequence aligning of the 16 inhibitor phages suggested that the sequences of inhibitor peptides obtained by the two separate screening methods appeared overlapping, and included a consensus motif PX(0-3)H/R or H/RX(0-3)R, in which X represented a non-specific amino acid.

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PubMed1997

The Interaction of Recombinant Androgen Receptor with the Androgen Responsive Element.

A 1 119 bp (1105 to 2224) fragment of androgen receptor (AR) cDNA, named AR1 (containing the whole DNA binding domain, the hinge site region and the partial hormone binding domain) was constructed into an expression vector pGEX. The GST-ARI fusion protein was expressed in E. coli induced by IPTG and purified from glutathione-Sepharose-4B affinity column. With a known ARE of the C3(l) gene as positive probe, the expressed product was proved to be AR with high ARE-binding affinity by gel shifting assay(EMSA) and in vitro Dnase I footprinting analysis.

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PubMed1997

His(6) Fusion Expression of Myristoyl-CoA: Protein N-myristoyltransferase in E. coli and its Purification.

Saccharomyces cerevisiae Myristoyl-CoA: protein N-myristoyltransferase (NMT) gene was cloned into a His(6)-fusion expression vector pMFHT. After transforming into E. coli BL21 (DE3), His(6)-NMT was induced to express at 37 degrees by IPTG. SDS-PAGE analysis showed an induced expression product band of about 54 kD which constituted about 10% of the total bacterial proteins. The analysis of product solubility revealed that His(6)-NMT was predominantly soluble. On the basis of these results, His(6)-NMT was purified in one-step to 95% of purity from bacterial lysates using immobilized metal (Ni(2+)) chelation affinity chromatography. The in vitro labelling experiment demonstrated that His(6)-NMT had an activity similar to that of mature NMT, suggesting that the His(6)-tag did not affect the enzyme activity. His(6)-tag in the N-terminal of NMT makes it be possible to immobilized simply on Ni(2+)-IDA Sepharose 6B resin, which can be used to screen the peptide inhibitors of NMT from Phage Display random peptide library.

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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

Cloning and Expression of the cDNA Coding for Rat Peptidylglycine alpha-Amidating Monooxygenase.

The dDNA of genes encoding rat peptidylglycine alpha-aminating monooxygenase(rPAM) were cloned and their expression in E. coli studied. Three DNA fragments were isolated from the rat brain cDNA library using the methods of plaque hybridization and PCR. DNA sequencing showed that they contained the total coding sequence for rPAM-2. By using the sited-directed mutation and PCR recombination methods, we obtained intact genes coding for the rPHM domain, the rPAL domain and rPAM, respectively. Different plasmids of these genes controlled under T(7) or P(L) promoter were constructed and transformed into E. coli. The high-level expression of rPAM-N260 in E. coli was first observed and its antiserum was prepared for the immunoassay of natural or recombinant PAMs. By the analysis with SDS-PAGE and Western blot, the products of rPHM and rPAM in E. coli were detected, and the amount of rPHM reached over 10% of the total bacterial proteins. It was found that low temperature and copper ion obviously increased the stability and solubility of the rPHM expressed in E. coli.

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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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PubMed1996

Expression of Human Bone Morphogenetic Protein 2 in Bombyx mori Larvae and Its Purification.

Recombinant Bm-BacPAK-BMP2 virus which contains the human bone morphogenetic protein 2 (BMP2) cDNA gene under the control of polyhedrin gene promoter was generated by cotransfection into Bm-N cells with the transfer plasmid pBacPAK-BMP2 and the modified Bombyx mori nuclear polyhedrosis virus Bm-BacPAK genomic DNA. On the fifth day after infection with the recombinant virus, Bombyx mori larvae produced about 10 &mgr;g rBMP2 in 1 ml hemolymph. The expressed rBMP2 was processed into 16 kD C-terminal fragments and formed 30 kD dimmers with disulfide bonds in vivo. The protein was purified to about 90% purity and induced cartilage locally in rats 7 days after subcutaneous transplantation.

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PubMed1996

Cloning and Expression of the Yeast Saccharomyces cerevisiae MyristoyL-CoA: Protein N-Myristoyltransferase Gene.

The gene encoding the myristoyl-CoA: protein N-myristoyltransferase from yeast S. cerevisiae (YSCNMT) has been amplified with the synthetic oligonucleotides by PCR, cloned into the pBluescript KS+ vector and analyzed by DNA sequencing. Then, an expression plasmid (pMFT7-5-NMT) containing the cloned YSCNMT gene controlled by T7 promoter has been constructed and transformed into E. coli BL21(DE3). Analyzed by SDS-PAGE, an IPTG-induced product with the same molecular size (53 kD) as that deduced from the known YSCNMT amino acid sequence was observed. It accounted for about 39% of the total cellular proteins, the soluble form of which accounted for about 34% of the total soluble cellular proteins and was purified to homogeneity in a single step using P11 phosphocellulose chromatography. The N-terminal amino acid sequencing revealed that the expressed protein began with the sequence encoded by the cloned YSCNMT gene and did not contain the N-terminal methionine. It was also observed that the expressed product had apparent YSCNMT activity in the in vitro assay.

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PubMed1996

Expression of C-terminal Segment of Bone Morphogenetic Protein in E. coli.

Two recombinant C-terminal segments of human bone morphogenetic protein 2A (BMP2A), designated as BMP23 (173 aa) and BMP24 (134 aa), were expressed under the control of P(L) promoter in E. coli. SDS-PAGE analysis showed two induced protein bands at 20 kD and 15.5 kD, which were about 10% and 20% of the total bacterial protein, respectively. Both the expressed proteins were insoluble aggregates, which reached 80% purity after rapid preparation of the products as the inclusion body. The results of N-terminal amino acid sequencing showed that the first 15amino acids at the N-terminal were identical to those encoded by the cDNA genes of both BMP24. After refolding both BMP23 and BMP24 formed disulfide-linked dimeric protein bands in SDS-PAGE. Bioassay in vivo revealed that BMP23 induced chondrocytes differentiation and BMP24 stimulated collagen synthesis.

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