Biomedical subjects

Li-Xin Ma

Publications and source records attributed to Li-Xin Ma.

9 recordsLinked to original sources

PubMed2006

Expression and purification of two lipases from Yarrowia lipolytica AS 2.1216.

We isolated two lipase genes LIPY7, LIPY8 from Yarrowia lipolytica CGMCC (China general microbiological culture collection center) AS 2.1216. The LIPY7 and LIPY8 genes encode a 366 and a 371-amino acid protein, respectively. The lipase genes with 6 x His tag sequence were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host Pichia pastoris KM71, respectively. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized.

Bacterial Proteins↗

PubMed2006

[A universal high-throughput novel method of constructing the vectors].

In this paper, a simple universal high-throughput method of constructing the vectors was developed. It could add proper adapter when the PCR primers were designed, and the purpose fragments were cloned by PCR, and the various complementary sticky ends were created by T4 DNA polymerase's 3' -exodeoxyribonuclease activity. If all these fragments were put together with DNA ligase, they would recombinate in an orientation. If they had been transformated, the tansformants would be identified. Let's take the Oryza sativa single-cross homologous recombination chloroplast expression vector pRSMGA which was constructed with seven fragments as an example, if the vector pRSMGA was constructed in using the method what had mentioned, only twice recombination and transformation would be done. Scores of experiments had proved that it is a simple universal high-throughput novel method to construct the complicated vectors, which has not appeared in the periodical.

Genetic Vectors↗

PubMed2006

[An optimized method for construction of genomic library].

Construction of genomic libraries is basic and important. Because of the laboriousness and high background of traditional methods for constructing genomic libraries, we improved them by overcoming these disadvantages. Two Ear I sites were chosen as the cloning sites, which can produce variable 3-base cohesive ends. Therefore the two overhangs could be devised to prevent a match and to avoid self-ligation of vector. Genomic DNA is cleaved partially with Sau3A I and subsequently incubated with dGTP and Klenow fragment of DNA polymerase Iso the self-ligation of fragments and ligation between them are blocked. In this study, the ARS probe vector (pHBM803/Trp) based on the improved method was constructed and then we constructed the Oryza sativa genomic library separately with the traditional method and improved method and compared them. The result of experiment indicated that the improved method could optimize the quality of library.

Cloning, Molecular↗

PubMed2005

[Cloning and expression in Pichia pastoris of an alkaline mannanase gene].

A strain containing alkaline mannanase gene was isolated from soil by functional plates and the genome library was constructed. From it a mannanase gene TM1 was acquired and was sequenced. The BLAST analysis showed a lower-than-60% similarity of the amino acid sequence to those in GenBank and proved TM1 to be a new mannanase gene (GenBank accession number AY623903). The new gene without signal peptide was cloned into the Pichia pastoris expression vector pHBM905C. The recombinant plasmid pHBM1201 was digested by Sal I and transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. All of the recombinant Pichia pastroris strains containing pHBM1201 secreted functional beta-mannanase. Because of its high mass of expression, the recombinant Pichia pastoris SMD1168-3 containing pHBM1201 was induced at shake flasks. The optimal temperature and pH of the beta-mannanase produced by the recombinant strains were 55 degrees C and 7.5, respectively. The enzymatic activity for konjak powder reached 41.8 with a half life of one hour. After keeping at 80 degrees C for 5 min, the enzymatic activity declined from 77% to 11% and the enzymatic activity could recover up to more than 60% when the temperature descended to 55 degrees C.

Cloning, Molecular↗

PubMed2005

[Expression of a laccase gene from Pleurotus ostreatus in Pichia pastoris and characterization of the recombinant enzyme].

A Pleuotus ostreatus laccase gene was cloned by RT-PCR and designated as lccPol. Its sequence was submitted to GenBank with the accession number AY450404 obtained. The open reading frame was transformed into three Pichia pastoris strains GS115, KM71 and SMD1168, respectively, under control of the AOX1 promoter by using the vector pHBM906. LCCPo1 can be expressed by all three P. pastoris recombinant strains. Three different strategies for shake-flask cultures were compared: (1) (25 degrees C, 1.0% methanol), (2) (20 degrees C, 1.0% methanol), (3) (20 degrees C, 0.5% methanol). The laccase activity could be improved by increasing the methanol concentration befittingly. The results showed that the cultivation temperature had a marked effect on the production of active heterologous laccase. 2 - 6 folds higher laccase activities were obtained when the cultivation temperature was kept at 20 degrees C instead of 25 degrees C. The highest activities, 3.19U/mL [GS115 (pHBM565)], 2.56U/mL [KM71 (pHBM565)], and 2.49U/mL [SMD1168 (pHBM565)], were gotten when the induction were performed at 20 degrees C with 1.0% (V/V) methanol supplied. The temperature and pH optimum for the recombinant laccase produced by three strains were 60 degrees C and pH4.2, respectively.

Base Sequence↗

PubMed2005

[Construction of tobacco chloroplast multicistron site integration expression vector and its transgene].

According to the published DNA sequence, a serial of elements for constructing the tobacco chloroplast multicistron site integrating expression vectors have been cloned by PCR technique, which include Prrn (a modified plastid ribosomal RNA operon promoter), psbA3' (the 3' region of the plastid psbA gene), aadA gene (encoding aminoglycoside 3'-adenylytransferase), man gene (encoding mannase), gfp gene (encoding green fluorescence protein) and tobacco chloroplast high-frequency homologous recombination ctDNA fragment (psaA/psbC, 3463 bp) (Fig.2). A tobacco chloroplast multicistron expression vector pLM4 (Fig.1) (-psaA-Prrn-SD-man-SD-gfp-SD-aadA-psbA3'- psbC-) was constructed with these elements. Then the tobacco leaves were bombarded 5 times with gold particles coated with the vector pLM4. After growing on the screening medium, the function of aadA gene was identified (Fig.3), and the function of gfp gene was confirmed by laser scanner (Fig.4), the expression of man was identified by Western blot (Fig.5). All these genes man, gfp and aadA being integrated in the tobacco chloroplast genome DNA were confirmed by PCR (Fig.6). And the multicistron expression cassette integrating in tobacco chloroplast genome DNA was confirmed by RFLP (Fig.7). All these showed that the three genes in the tomato vector pLM4 were expressed in tobacco chloroplast genome DNA.

Binding Sites↗

PubMed2004

[Cloning of a laccase gene from Flammulina velutipes and study on its expression in Pichia pastoris].

Laccase(EC1.10.3.2) can be used for enzymatic detoxification of lignocellulosic hydrolysates. By using molecular techniques such as RACE (rapid amplification of cDNA ends) and Genome-Walking, a laccase gene and its corresponding full-length cDNA were cloned from Flammulina velutipes and designated as glccFv and IccFv. The sequences were submitted to GenBank, and the accession numbers obtained were AY485826 and AY450406, respectively. Analysis of amino acids sequence suggested that one laccase from Polyporus ciliatus possessed the highest homology with the protein encoded by lccFv showing for 72%. The ORF (open reading frame) of lccFv was transformed into Pichia pastoris strain GS115 through the P. pastoris expression vector pHBM906, which contains both the promoter and transcription terminator of the AOX1 gene. The recombinant laccase LCCFv was detected from the engineering strain GS115 (pHBM557) which was fermented with BMMY liquid medium and induced by 1.0% (V/V) methanol at 20 degrees C with the highest expression level (0.1070 U/mL). The optimal reaction temperature of LCCFv that secreted from P. pastoris GS115(pHBM557) was 45 degrees C, the optimal reaction pH value was pH3.9 and the thermostability and pH stability were very well under the optimal conditions.

Base Sequence↗

PubMed2003

[Secreted expression of Bacillus pumilus xylanase gene in Pichia pastoris and study on enzymatic properties].

The endo-1,4-xylanase gene from Bacillus pumilus HB030 was cloned into the Pichia pastoris expression vector, pPIC9k, the recombinant plasmid was named pHBM220. The digested recombinant plasmid pHBM220 was transformed into Pichia pastoris KM71, GS115, SMD1168, respectively. The recombinant Pichia pastoris KM71 (pHBM220), GS115 (pHBM220), SMD1168 (pHBM220) secreted functional endo-1,4-xylanase, and the enzymatic activities reached 10.80IU/mL, 11.63IU/mL, 9.68IU/mL, respectively. The temperature and pH optimum for the recombinant xylanase were 60 degrees C and pH5.5, respectively.

Bacillus↗

PubMed2003

[Expression and identification of recombinant arresten in Pichia pastoris].

Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.

Arrestin↗