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fusion expression of Glucoamylase and Xylanase in Pichia pastoris

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Agricultural Biotechnology 2012年第5期1卷 48-51页

作者： Zhi WANG Baoqing DUN Jingang GU Xuan ZHAO Gu TIAN Ming LU Guiying LI National Key Facility for Crop Gene Resources and Genetic Improvement of Institute of Crop Sciences Chinese Academy of Agricultural Sciences Agricultural Culture Collection of China Institute of Agricultural Resources and Agricultural DivisionsChinese Academy of Agricultural Sciences College of Life Sciences Northeast Agricultural University

Employing RT-PCR amplification method, the mature pepfide coding sequence of glucoamylase （amyA） gene was amplified from total RNA of Talaro- myces emersonii and the xylanase （xyrut） gene from genomic DNA ofPaenibacillus sp. H10-3. The result showed that the mature peptide sequence of amyA is 1857 bp long and encodes 618 amino acids; and the mature peptide sequence of xynA is 636 bp long and encodes 211 amino acids. Then these two genes were spliced by overlap extension PCR （ SOE-PCR）, yielding fusion gene amyA-l-xynA. The fusion gene amyA-l-xynA was then cloned into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid pPIC9-amyA-l-xynA. The recombinant plasmid pPICg-amyA-l-xynA was linearized and transformed into Pichia pastoris GSll5 via electric shock, yielding engineering strain ALX2. The maximum yields of glucoamylase and xylanase in ALX2 fermentation supernatant were determined as 10.7 and 51.8 U/ml, respectively.

关键词： Glucoamylase Xylanase fusion expression Pichia pastoris

fusion expression of immunological epitopes from VP1-3 of foot-and-mouth disease virus with porcine interferon-gamma

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Fusion expression of immunological epitopes from VP1-3 of fo...

Eukaryotic Secrete fusion expression of Rhizopus oryzae glucoamylase and Paenibacillus ***

2006年度学术研讨会

作者： YAO Qing-xia, QIAN Ping, CAO Yi, XU Zhuo-fei, HE Yan-nan, SI You-hui, CHEN Huan-chun (Laboratory of Animal Virology, College of Animal Science and Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei Province, 430070, China)

The artificial synthetic immunological epitopes from VP1-3 of foot-and-mouth disease virus (FMDV) were inserted into prokaryotic expression vector (pGEX-KG) in cascade connection with porcine interferon-γ. The recombinant plasmids were identified by enzymes and sequencing. The identified recombinant plasmid was transformed into BL21 and induced by IPTG to express the fusion protein in high quantities. The expressed product was analyzed by SDS-PAGE, and the results demonstrated that the molecular weight of it was about 66 kDa, which was consisted with the anticipation. And the interest protein was about 37.4% of the total protein by scanning, which mainly existed in inclusion bodies. After these inclusion bodies were disnatured by SKL, they were renatured by PEG4000, oxidation tathion and reduction tathion. The crude extracted product was analyzed by Western-blotting, which indicated that the expressed protein had the biologic activity. This study established foundation to the next work of genetically engineered vaccines.

关键词： foot-and-mouth disease virus antigen epitope porcine interferon-γ fusion expression

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Eukaryotic Secrete fusion expression of Rhizopus oryzae gluc...

expression of Mouse MT-1 as a fusion Protein in Anabaena sp. PCC 7120

第五届全国微生物资源学术暨国家微生物资源平台运行服务研讨会

作者： Wang Zhi Dun Bao-Qing Lu Ming The National Key Facility for Crop Gene Resources and Genetic Improvement of Institute of Crop Sciences Chinese Academy of Agricultural Sciences

The glucoamylase gene(amy A) was amplified from Rhizopus oryzae 3.866 total DNA extracts. The result showed that the sequence of amyA was consisted of 2049 bp,and encoded 604 amino *** xylanase gene(xynA) was amplified from Paenibacillus sp.H10-3 total DNA *** result showed that the mature peptide sequence of xynA was consisted of 636bp,and encoded 211 amino ***,the mature peptide sequence of amyA gene,sequence of linker and sequence of xyn A gene were spliced by overlap extension PCR(SOE-PCR),which amyA-l-xynA was *** fusion gene was then inserted into a Pichia pastoris expression vector pPIC9 to produce the recombinant expression plasmid p?*** using electric shocks,we successfully transformed the recombinant pP\C9-amyA-l-xynA into Pichia pastoris GS115,which AX 11 was obtained..The maximum yield of the recombinant glucoamylase and xylanase in AX11 culture medium were 5.8U/mL and 32.3U/mL, respectively.

关键词： glucoamylase xylanase fusion expression Pichia pastoris

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Acta Botanica Sinica 2003年第1期45卷 98-101页

作者：周杰郝福英施定基俞梅敏茹炳根北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室北京100871 中国科学院植物研究所北京100093

To produce mouse metallothionein-1 (mMT-I ) in cyanobacterium Anabaena sp. PCC 7120, a novel Escherichia co/i-cyanobacterium shuttle fusion expression vector, pKG-MT, was constructed. Via this vector, mMT-I cDNA which was fused with a carboxyl terminal extension of the 26 kD glutathione-S-trans-ferase (GST) containing a thrombin specific site was expressed in Anabaena under the control of tac promoter. SDS-polyacrylamid gel electrophoresis (SDS-PAGE) showed that the fusion protein GST-MT was expressed in the transgenic Anabaena sp. PCC 7120 after induction with isopropylthio-β-D-galactoside (IPTG). Glutatione-S-transferase metallothionein (GST-MT) was purified from the crude extracts by affinity chromatography on immobilized glutathione and mMT- I was obtained by digesting the fusion protein with thrombin on column and gel filtration on Sephadex G-50. SDS-PAGE demonstrated that the purified mMT- I was the desired protein. The result of ELISA for the purified mMT-I showed that the recovery of mMT- I from the transgenic cyanobacterium was about 0.6 mg/g fresh weight. According to the data of atomic absorption assay, metal-binding activity of the purified mMT-I was almost the same as that of wild type MT.

关键词： mouse metallothionein-I Anabaena sp. PCC 7120 fusion expression affinity chromatography

Overexpression of Soluble Human Thymosin Alpha 1 in Escherichia coli

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Acta Biochimica et Biophysica Sinica 2005年第2期37卷 147-151页

作者： Pei-Fu CHEN Hong-Ying ZHANG Geng-Feng FU Gen-Xing XU Ya-Yi HOU Medical School,Nanjing University.Nanjing 210093,China School of Life Sciences.Nanjing University.Nanjing 210093,China Medical School Nanjing University Nanjing 210093 China School of Life Sciences Nanjing University Nanjing 210093 China

Synthesized gene of human thymosin alpha 1（Tα1）was inserted into pET-28a,pET-9c,pThioHis ***-2T or pBV222 and then inductively expressed in strains of Escherichia *** thefive expression systems,the BL21/pET-28a system provides the highest expression level of fusion protein ina soluble form,which is up to 70% of total expressed bacterial proteins as visualized by sodium dodecylsulfate-polyacrylamide gel electrophoresis（SDS-PAGE）.The resulting fusion protein purified through nickelaffinity chromatography accounts for 2.53% of the wet bacterial pellet weight and reaches 94.5% purity *** results indicate the potential of this expression system for high-throughput production ofrecombinant Tα1.

关键词： human thymosin alpha l Escherichia coli fusion expression Ni2+ affinity chromatography

Engineering Saccharomyces cerevisiae for enhanced(–)-α-bisabolol production

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Synthetic and Systems Biotechnology 2023年第2期8卷 187-195页

作者： Yinkun Jiang Lu Xia Song Gao Ning Li Shiqin Yu Jingwen Zhou Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology Jiangnan University1800 Lihu RoadWuxiJiangsu214122China Science Center for Future Foods Jiangnan University1800 Lihu RoadWuxiJiangsu214122China Key Laboratory of Industrial Biotechnology Ministry of Education and School of BiotechnologyJiangnan University1800 Lihu RoadWuxiJiangsu214122China Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology Jiangnan UniversityWuxi214122China

(–)-α-Bisabolol is naturally occurring in many plants and has great potential in health products and ***,the current extraction method from natural plants is unsustainable and cannot fulfil the increasing *** study aimed to develop a sustainable strategy to enhance the biosynthesis of(–)-α-bisabolol by metabolic *** introducing the heterologous gene MrBBS and weakening the competitive pathway gene ERG9,a de novo(–)-α-bisabolol biosynthesis strain was constructed that could produce 221.96 mg/L(–)-α-*** key genes for(–)-α-bisabolol biosynthesis,ERG20 and MrBBS,were fused by a flexible linker(GGGS)3 under the GAL7 promoter control,and the titer was increased by *** of the mevalonic acid pathway and multi-copy integration further increased(–)-α-bisabolol *** promote product efflux,overexpression of PDR15 led to an increase in extracellular *** with the optimal strategy,(–)-α-bisabolol production in a 5 L bioreactor reached 7.02 g/L,which is the highest titer reported in yeast to *** work provides a reference for the efficient production of(–)-α-bisabolol in yeast.

关键词： Metabolic engineering (–)-α-bisabolol fusion expression Transporter Saccharomyces cerevisiae

Prokaryotic expression of Antimicrobial Peptide CATH PR1–2 from the Skin of Paa robertingeri in Escherichia coli

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Asian Herpetological Research 2017年第4期18卷 275-283页

作者： Huaiqing DENG Chen CHEN Ning XIAO Jiang ZHOU College of Life Sciences Guizhou Normal University Guiyang 550001 Guizhou China

The aim of this study was to investigate the prokaryotic expression of antimicrobial peptide cathelicidin （CATH） PR1 and PR2 from the skin of Paa robertingeri in Escherichia coli. Two active peptides, CATH PR1 and CATH PR2, belong to the CATH family in the skin of P. robertingeri. CATH PR1 has a relatively high antimicrobial activity, especially for the drug-resistant strains found in clinical practice; however, no antimicrobial activity has been found in CATH PR2. The molecular weights of both CATH PR1 and CATH PR2 are relatively low （3195.88 and 2838.34 Da, respectively）. Thus, the genetic processes, as well as the expression and purification of these proteins, are difficult to perform. Therefore, in this study, CATH PR1 and CATH PR2 genes were tandem ligated and then connected to the plasmid pET-32a. This reconstructed plasmid was then transfected into the expression vector E. coli BL21 to construct the recombinant expression system. The fusion expression of peptide PR was stable in E. coli after induction with 1.0 mol/L isopropyl β-D-1-thiogalactopyranoside at 37℃ for 4 h. The antimicrobial activity assay using Staphylococcus aureus （Song） and Candida albicans 08030102 showed that the antimicrobial activity of PR was similar to the antimicrobial activity of CATH PR1. This study showed that artificial modification of the amino acid sequences of PR1 and PR2 could result in better protein expression in prokaryotes, and the fusion protein expressed had relatively high antimicrobial and other biological activities. In conclusion, the findings suggest future prospects of the commercialization of this method.

关键词： E. coli BL21 fusion expression Paa robertingeri recombinant protein PR

Optimization of the Purification Methods for Recovery of Recombinant Growth Hormone from Paralichthys olivaceus

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Journal of Ocean University of China 2013年第1期12卷 169-174页

作者： ZANG Xiaonan ZHANG Xuecheng MU Xiaosheng LIU Bin College of Marine Life Sciences Ocean University of China Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone （r-fGH） was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield （2.605 mg L^-1） than that from soluble protein （1.964 mg L^-l）. Of the tested recovery methods, addition of renaturing buffer （pH 8.5） into denatured inclusion body yielded the best recovery rate （17.9%）. This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.

关键词： Paralichthys olivaceus growth hormone fusion expression purification receptor binding activity

Gene Cloning and expression of PKA Catalytic Subunit and Its Activity in Arabidopsis thaliana

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Acta Botanica Sinica 2003年第4期45卷 460-465页

作者：刘璞陈珈张会霞王学臣中国农业大学生物学院植物生理生化国家重点实验室北京100094

Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.

关键词： cAMP-dependent protein kinase fusion expression activity assay