共查询到20条相似文献,搜索用时 15 毫秒
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High-level expression of a polypeptides encoded by a large segment of the envelope gene of HIV-1 in E. coli by directed evolution 总被引:1,自引:0,他引:1
Yongshu Zhang Zhihu Zhao Fuguang Li Cheng Cao Chuanxuan Liu Qingjun Ma 《Biotechnology letters》2000,22(11):947-950
A random mutagenesis library of gp120-801 (a large segment of the envelope protein gene of HIV-1) was constructed using error-prone PCR and DNA shuffling methods, and one mutant of gp120-801 was selected from this library using a green fluorescent protein (GFP) fusion vector. After being cloned into pEX31b and expressed in E. coli, the expressed fusion protein reached to 15% of total bacterial proteins for the mutant but was just 1–2% for the wild type. 相似文献
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Trichloroethylene (TCE) degradation by the recombinant E. coli JM109 harboring a TCE-degradative plasmid (pIO720 or pIO72K) in continuous culture was studied. The ampicillin-resistant plasmid, pIO720, contained the cumene dioxygenase genes and the dimethyl sulfide monooxygenase genes. pIO72K was constructed according to replacement of an ampicillin resistance gene on pIO720 by a kanamycin resistance gene. In the case of E. coli JM109 (pIO720) in continuous culture, TCE degradation activity decreased rapidly after continuous culture started, and the remaining number of host cells harboring pIO720 also decreased rapidly. In the case of E. coli JM109 (pIO72K) in continuous culture, TCE degradation activity was stable during continuous culture for at least 300 h and the number of the host cells harboring pIO72K did not decrease. TCE degradation activity of E. coli JM109 (pIO72K) was the highest at a dilution rate of 0.2 h–1. 相似文献
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Kim M Kwon T Lee HJ Kim KH Chung DK Ji GE Byeon ES Lee JH 《Biotechnology letters》2003,25(15):1211-1217
A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl--d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity. 相似文献
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Recombinant bovine angiogenin (rbAng) was expressed in E. coli at up to 30% of total cell proteins but was produced as inclusion bodies. By investigating the effect of various factors on the refolding yield, we obtained about 60% refolding. After chromatographic purification, about 60 mg purified angiogenin was obtained from 1 l culture. The purified recombinant bovine angiogenin was identical to native bovine angiogenin (nbAng) obtained from cow's milk. Our approach is highly efficient and can be generally used for the production of various types of angiogenin for functional and structural studies as well as therapeutic purposes. 相似文献
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Qian Jia HongTao Wu XingJun Zhou Jian Gao Wei Zhao JouDi Aziz JingShuang Wei Lihua Hou Shuyin Wu Ying Zhang XiangFeng Dong YanMin Huang WeiYuan Jin HongJie Zhu XinHui Zhao ChunHua Huang LiPing Xing Liwen Li Jun Ma Xiyan Liu Ran Tao ShuaiDong Ye YiGao Song LingLing Song GuanPing Chen ChunLing Du XueTing Zhang Bo Li YanTao Wang Wei Yang Gilbert Rishton YuYang Teng GouQing Leng LuanFeng Li WenXian Liu LiJun Cheng QiuBo Liang ZhengWu Li XiuQin Zhang Yajun Zuo Wei Chen Huicheng Li Matthew Hui 《中国科学:生命科学英文版》2010,53(1):94-100
High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein. The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment (including intron) of extremely GC-rich at the 5′ or/and 3′ flanking regions of these protein genes or their gene promoters. This experiment is the first experimental evidence showing that a non-coding extremely GC-rich DNA fragment is a super “chromatin opening element” and plays an important role in mammalian gene expression. This experiment has further indicated that chromatin-based regulation of mammalian gene expression is at least partially embedded in DNA primary structure, namely DNA GC-content. 相似文献
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Soltanian Bita Dehghan Shasaltaneh Marzieh Riazi Gholam Hossein Masoudian Nahid 《Molecular biology reports》2021,48(8):6103-6112
Molecular Biology Reports - Alzheimer's disease (AD) is a degenerative brain disorder. Due to the relationship between the functional loss of astrocytes and AD, the present study aims to... 相似文献
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High mammalian gene expression was obtained for more than twenty different proteins in different cell types by just a few laboratory scale stable gene transfections for each protein.The stable expression vectors were constructed by inserting a naturally-occurring 1.006 kb or a synthetic 0.733 kb DNA fragment(including intron) of extremely GC-rich at the 5' or/and 3' flanking regions of these protein genes or their gene promoters.This experiment is the first experimental evidence showing that a non-coding ex... 相似文献
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Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions. 相似文献
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Mirkin S. M. Zaitsev E. N. Panyutin I. G. Lyamichev V. I. 《Molecular genetics and genomics : MGG》1984,194(3):508-512
Molecular Genetics and Genomics - This study deals with the effects of a temperature-sensitive (ts) mutation at the gene encoding the DNA gyrase B subunit (gyrB ts) and a deletion of the top gene... 相似文献
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Niranjali Gamage Takanori Matsuura Naoko Norioka Yumi Yoshimura Takeshi Takasaki Tetsu Nakanishi Shigemi Norioka 《Biotechnology letters》2000,22(17):1413-1417
S-RNase is a style-specific ribonuclease which is associated with gametophytic self-incompatibility. An expression vector of a fusion protein of Pyrus pyrifolia(Japanese pear) S3-RNase with glutathione-S-transferase (GST) was constructed and transformed into E. coli. Using this system, the fusion protein, GST-S3-RNase, was expressed as an active form and can be used for screening pollen S-gene product(s). 相似文献
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Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli. 相似文献