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Bo-Liang Li

Publications and source records attributed to Bo-Liang Li.

At least 19 recordsLinked to original sources
PubMed2004

Human acyl-coenzyme A:cholesterol acyltransferase 1 (acat1) sequences located in two different chromosomes (7 and 1) are required to produce a novel ACAT1 isoenzyme with additional sequence at the N terminus.

A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

Amino Acid Sequence↗
PubMed2004

Functional contribution of EEN to leukemogenic transformation by MLL-EEN fusion protein.

The EEN (extra eleven nineteen) gene was originally cloned from a case of acute myeloid leukemia M5 subtype with translocation t (11; 19)(q23; p13), in which EEN was fused with MLL. To explore the involvement of EEN in leukemogenesis caused by MLL-EEN, we studied the transformation potential of the MLL-EEN fusion protein. MLL-EEN had oncogenic features, while, as a control, MLLDelta, the truncated form of MLL lacking the EEN moiety, did not show any oncogenic potential. MLL-EEN exerted a dominant-negative effect over wild-type EEN in terms of subcellular localization. Normally, EEN was found in the cytoplasm, but the MLL-EEN fusion protein was located in the nucleus, and EEN could be delocalized by MLL-EEN. This interaction is via a coiled-coil dimerization domain of EEN, which is reserved in the fusion protein. In addition, MLL-EEN might act as a potential transcriptional factor with the MLL part providing the DNA-binding domain and the EEN part providing the transcription activation domain, though EEN seems to have no direct role in transcriptional regulation. As an aberrant transcriptional factor, MLL-EEN could transactivate the promoter of HoxA7, a potential target gene of MLL.

Amino Acid Sequence↗
PubMed2004

A stable upstream stem-loop structure enhances selection of the first 5'-ORF-AUG as a main start codon for translation initiation of human ACAT1 mRNA.

Human ACAT 1 cDNA K1 was first cloned and functionally expressed in 1993. There are two adjacent in-frame AUG codons, AUG(1397-1399) and AUG(1415-1417), at 5'-terminus of the open reading frame (ORF, nt 1397-3049) of human ACAT1 mRNA corresponding to cDNA K1. In current work, these two adjacent inframe AUGs at 5'-terminus of the predicted ORF (5'-ORF-AUGs) as start codons for translation initiation of human ACAT1 mRNA were characterized in detail. Codon mutations indicated that both of these two adjacent 5'-ORF-AUGs can be selected as start codons but the first 5'-ORF-AUG(1397-1399) is a main start codon consistent with that of the predicted ORF of human ACAT1 mRNA. Further deletion and mutation analyses demonstrated that a stable upstream stem-loop structure enhanced the selection of the first 5'-ORF-AUG(1397 -1399) as a main start codon, in addition to upstream nucleotide A in the -3 position, which is a key site of Kozak sequence. In addition, result of ACAT1 enzymatic activity assay showed no obvious difference between these two ACAT1 proteins respectively initiated from the two adjacent 5'-ORF-AUGs. This work showed that a stable upstream stem-loop structure could modulate the start codon selection during translation initiation of mRNAs that contain adjacent multi-5'-ORF-AUGs.

5' Untranslated Regions↗
PubMed2003

Bacterial expression, purification, and in vitro N-myristoylation of fusion hepatitis B virus preS1 with the native-type N-terminus.

Very low-level expression of hepatitis B virus (HBV) preS1 with the native-type N-terminus hampered the biochemical and functional studies on its myristoylation. In the present study, the fusion HBV preS1 with the native-type N-terminus and a His6-Tag fused to C-terminus (HBV preS1-HT) was highly expressed in Escherichia coli. This was due to an introduced mutation of the rare codon GGA found in the HBV preS1 to the codon preferred by E. coli, GGU. The protein was rapidly purified from bacterial lysate by Ni-IDA affinity chromatography. The experimental assays using 3H-labeled substrate demonstrate that the purified HBV preS1-HT can be effectively N-myristoylated by recombinant human protein N-myristoyltransferase (NMT) in vitro.

Acyltransferases↗
PubMed2003

Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter.

The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.

Amino Acid Sequence↗
PubMed2003

Expression, purification and in vitro N-myristoylation of human Src N-terminal region.

The DNA fragment encoding N-terminal region of human c-Src was amplified from Caco-2 cell total RNA by RT-PCR and cloned into vector pMFHT to obtain His-tag fusion expression plasmid pMF-SrcHT, which was based on T7 expression system. The fusion protein SrcHT was highly expressed in E.coli BL21(DE3) harboring the pMF-SrcHT and purified from bacterial lysate by Ni-IDA affinity chromatography. The assays using [(3)H]-labeled substrate demonstrate that the purified fusion protein SrcHT can be effectively N-myristoylated by recombinant human myristoyl-CoA: protein N-myristoyltransferase (NMT) in vitro. This work is a basis for further biochemical studies and development of new anti-cancer chemotherapeutic drugs based on specific inhibition of N-myristoylation of human Src.

Amino Acid Sequence↗
PubMed2002

[Couple production of human calcitonin and rat peptidylglycine alpha-amidation monooxygenase in insect cells].

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.

Animals↗
PubMed2000

In Vitro Amidating Processing of Products Expressed by Gene Engineering.

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.

Journal Article↗
PubMed1999

Synthesis and Biological Activity of Human Calcitonin Analogue.

The potency of salmon calcitonin (sCT) is higher but the structural homology between sCT and human calcition (hCT) is only 50%. Based on the comparison of the structure between sCT and hCT, we have designed and synthesized a hCT analogue (mhCT-2) by solid phase method, using air oxidation in diluted solution to obtain a peptide with intramolecular disulfide bond. Through HPLC purification, we obtained a capillary electrophoresis-homogeneous mhCT, results of analysis of its mass spectrum and N-terminal sequencing were in accordance with the theoretical values. The results of calcitonin bioassay by estimating the blood calcium levels in rats showed that the potency of mhCT-2 was around 2 000 IU/mg, one order of magnitude higher than that of hCT. In RIA assay, we have found that the immunoactivity of mhCT-2 and hCT was much different because of their different binding abilities to anti-hCT antibody. This indicated that the conformation of mhCT-2 was changed as compared with hCT. In rat osteoporosis model, the results showed that pharmacologic effects of mhCT-2 was the same as that of sCT. The synthetic mhCT-2 seems promising to be a clinically useful peptide with high potential in osteoporosis therapy, because it is similar in biological properties to, but less immunogenic than sCT.

Journal Article↗
PubMed1999

Expression Purification and Activity ofhuman Myristoyl-CoA:N-myristoyltransferase.

A gene encodinghuman myristoyl-CoA: protein N-myristoyltransferase (hNMT) from a brain cDNA libraryhas been obtained by PCR amplification and DNA sequencing. Then the mature-type and His(6)-fusion-type expression plasmids (pMF-hNMT(3) and pMFHT-hNMT(2)) containing the hNMT gene under control of T7 promoter have been constructed and transformed into E. coli BL21(DE3). SDS-PAGE analysis showed that the recombinant hNMT products expressed at 37 degrees were almost unsoluble but most of the His(6)-hNMT product expressed at lower temperature (22 degrees ) was soluble and its yield was about 7% of the total soluble cellular proteins. By immobilized metal (Ni(2+)) chelation affinity chromatography up to 80% His(6)-hNMT was purified by one step from bacterial lysate. The labelling experiment in vitro domonstrated that the expressed and purified His(6)-hNMT had an obvious catalytic activity to transferring myristoyl group.

Journal Article↗
PubMed1999

Efficient Secretion of Proteins Expressed from Insect Cells Directed by PAM Signal Peptide.

Signal and leading peptide sequences of rat PAM was inserted into the baculovirus transfer vector, and secretion expression plasmids pBACPAG2 and pBacPAI for the fusion gene PABC-hGRF and PABC-IGF-I were constructed, respectively. By cotransfection with linear genomic DNA of modified Autographa californica nuclear polyhedrosis virus (BacPAK6) and homologous recombination, the recombinant AcNPV, BacPAG and BacPAI, were obtained and identified. Fusion proteins PABC-hGRF and PABC-IGF-I were secreted efficiently from Sf21 cells infected with BacPAG and BacPAI, respectively, and those fusion protein could be purified efficiently by IgG affinity column.

Journal Article↗
PubMed1998

Studies on Structure and Function of the Myristoyltransferase Inhibitor Peptide Displayed on Phage Surface.

Site-directed mutagenesis was used to identify the critical residues and functional sequences of a potent NMT inhibitor peptide displayed on phage surface. W2A, H6N and H6R largely lowered the inhibitory activities of the mutated phages, and the activity of P3A and V4V5 right curved arrow A4A5 was also slightly reduced. However, V4V5 right curved arrow W4W5, H12N, H14N, C9S, C15S, deltaC15, deltaH14C15 and deltaC9--C15 had no obvious effect on the inhibition. These results suggest that W2, P3 and H6 are important in the peptide structure and/or the interaction with NMT, that amino acids with larger side chain are the more favorable residues between P3 and H6, and also that the sequences between C9 and C15 have no clear contribution to the inhibitory activity. In summary, the functional prptide directly related to the role of inhibition has been located between the N terminal T1 and A8 of the inhibitor peptide.

Journal Article↗
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.

Journal Article↗
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.

Journal Article↗
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.

Journal Article↗
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.

Journal Article↗