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Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

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Food Science and Human Wellness 2024年第4期13卷 2310-2316页

作者： Yuqi Zhu Pengdong Sun Chunjian Li Yu Zhang Yu Wang Jingyuan Li Yanfeng Liu Jian Chen Yang Deng College of Food Science and Engineering Qingdao Agricultural UniversityQingdao 266109China Key Laboratory of Special Food Processing(Co-construction by Ministry and Province) Ministry of Agriculture Rural AffairsQingdao Agricultural UniversityQingdao 266109China Qingdao Special Food Research Institute Qingdao 266109China Science Center for Future Foods Engineering Research Center of Ministry of Education on Food Synthetic BiotechnologyJiangsu Province Engineering Research Center of Food Synthetic BiotechnologyJiangnan UniversityWuxi 214122China Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan UniversityWuxi 214122China

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of *** this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-*** was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation *** original strain most likely only producedα-LA intracellular,but not *** improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the *** 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in *** YjcN was determined to be the best signal *** in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal *** addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and *** results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/*** study demonstrates successful expression and secretion of humanα-LA in *** and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

关键词： Bacillus subtilis Alpha-lactalbumin Bioengineering milk signal peptide Promoter screening

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signal peptide of Potato PinII Enhances the Expression of Cry1Ac in Transgenic Tobacco

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Acta Biochimica et Biophysica Sinica 2004年第8期36卷 553-558页

作者： Yun-Jun LIU, Yuan YUAN, Jun ZHENG, Ya-Zhong TAO, Zhi-Gang DONG,Jian-Hua WANG, and Guo-Ying WANGState Key Laboratory for Agrobiotechnology and National Center for Maize Improvement, China Agricultural University, Beijing 100094, China State Key Laboratory for Agrobiotechnology and National Center for Maize Improvement China Agricultural UniversityBeijing 100094 China

The modified Cry1Ac was expressed in transgenic tobacco plants. To allow secretion of the Cry1Ac protein into the intercellular space, the signal peptide sequence of potato proteinase inhibitor II (pinII) was N-terminally fused to the Cry1Ac encoding region. Expression of Cry1Ac in transgenic tobacco plants was assayed with ELISA. The results showed that pinII signal peptide sequence enhanced the expression of Cry1Ac protein and led to the secretion of the Cry1Ac protein in transgenic tobacco plants. GFP gene was also fused to the signal peptide sequence and transformed to tobacco. The results of fluorescent detection showed that GFP had localized in the apoplast of transgenic plants.

关键词： Cry1Ac signal peptide GFP transgenic tobacco

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signal peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2012年第2期32卷 299-302页

作者：王辰允王颖莹王淼陈建奎于农宋世平 Norbert E.KAMINSKI 张伟 Department of Pharmaceutical Administration PLA General Hospital Outpatient Department of Xiyuan PLA General Hospital Department of Clinical Laboratory No. 307 Hospital Academy of Military Medical Sciences Department of Pharmacology and Toxicology and the Center for Integrative Toxicology Michigan State University East Lansing

Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e.,≥95°C), forming a large molecular weight band when analyzed by immuno-blotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immuno-blot analysis. These findings provide very useful information for efficient mammalian expression and immuno-blotting of membrane receptors.

关键词： cannabinoid receptor 1 cannabinoid receptor 2 denaturing temperature signal peptide mammalian expression

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Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

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Journal of Central South University 2015年第6期22卷 2088-2095页

作者：王冶郑甲林福来周洪波 School of Minerals Processing and Bioengineering Central South University Key Laboratory of Biometallurgy of Ministry of Education (Central South University)

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

关键词： endo-1,4-β-mannosidase Pichia pastoris (P.pastoris) signal peptide optimization

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Structural Analysis of Fibroin Heavy Chain signal peptide of Silkworm Bombyx mori

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Acta Biochimica et Biophysica Sinica 2006年第7期38卷 507-513页

作者： Sheng-Peng WANG~(1,2)Ting-Qing GUO~1 Xiu-Yang GUO~1 Jun-Ting HUANG~2 Chang-De LU~(1*) 1 Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China 2 Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang 212018,China Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200031 China Sericultural Research Institute Chinese Academy of Agricultural SciencesZhenjiang 212018 China

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the *** effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a *** of EGFP fusion proteinswas examined under fluorescence *** SPs with lengths of 20,18,16 and 12 *** directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within *** *** the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk *** cleavage site of the SP is discussed andcompared with the predictive results of the signalP 3.0 online prediction program.

关键词： signal peptide fibroin heavy chain silkworm Bombyx mori rAcMNPV

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Computational Analysis of signal peptide-Dependent Secreted Proteins in Saccharomyces cerevisiae

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Agricultural Sciences in China 2006年第3期5卷 221-227页

作者： YANG Jing LI Cheng-yun WANG Yun-yue ZHU You-yong LI Jin-bin HE Xia-hong ZHOU Xiao-gang LIU Lin YE Yan-fen Key Laboratory for Agricultural Biodiversity for Pest Management Ministry of Education/~unnan Agricultural University Kunming 650223 P.R.China Plant Protection Research Institute Yunnan Academy of Agricultural Sciences Kunming 650201 P.R.China Biotechnology Research Institute Yunnan Academy of Agricultural Sciences Kunming 650223 P.R.China

Computer based software such as the signalP v3.0, TargetP v1.01, big-PI predictor and TMHMM v2.0 were combined to predict the signal peptides, and the signal peptide-dependent secreted proteins among the 6 700 ORFs in genome of Saccharomyces cerevisiae. The results showed that 163 proteins were the secreted ones containing signal peptides, and they were secreted via Sec pathway. Among the 163 predicted secreted proteins, the signal peptides of 47 secreted proteins included only the H-domain and C-domain, without N-domain, but the signal peptides of other 116 secreted proteins included all the three domains. There were differences in the constitution of signal peptides between the secreted proteins of S. cerevisiae and of Candida albicans, but the length and amino acids types of their signal peptides were similar in general. Few of the same signal peptides occurred in the secreted proteins of S. cerevisiae genome, and the homology could be compared among the secreted proteins with the same signal peptides. The BLAST 2 SEQUENECES and CLUSTAL W were used to align the two protein sequences and multi-protein sequences, respectively. The alignment result indicated that homology of these sequences with the same signal peptide was very highly conservative in amino acid of complete gene. The effect of the signal peptides in S. cerevisia on expression of foreign eukaryotic secreted proteins is discussed in this paper.

关键词： Saccharomyces cerevisia secreted protein signal peptide secretory pathway

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The Uncleaved signal peptide Determines the Nonclassical N-glycosylation of BmNPV GP64

The Uncleaved Signal Peptide Determines the Nonclassical N-g...

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中国蚕学会2023年学术年会

作者： Ying Xu Luping Sun Mengmeng Li Stephen Baffour Gyawu Bifang Hao Jinshan Huang

GP64,the major membrane fusion protein on budded virus(BV) of Bombyx mori nucleopolyhedrovirus(BmNPV),retains a signal peptide sequence in the mature virion,which determines the specific dependence on cholesterol and the virulence of BmNPV during *** it has been confirmed that the uncleaved signal peptide is crucial for GP64 to cross the plasma membrane,many other properties of GP64 have not been clarified *** order to further explore the effect of uncleaved signal peptide on expression,secretion,and modification,the N-glycosylation of GP64 and signal peptide-cleaved GP64(SPΔnGP64) were *** predicted N-glycosylation sites in GP64 were mutated(N→Q),and the expression,fusion activity,and infectivity were detected after the construction of mutant recombinant *** GP64 contains six potential N-linked glycosylation sites matching the consensus N-X-S/T motif,located at amino acids(AA) 5,175,213,370,400 and ***-directed mutagenesis was used to inactivate these predicted sites in GP64 and SPΔnGP64(lacking the 5th site in the SP n-region).Results showed that in GP64 mutants,N175Q and N442Q mutations eliminated virus infectivity and membrane fusion,while other single mutations only reduced infectivity and *** contrast,all SPΔnGP64 mutants were noninfectious,indicating all five sites were ***,liquid chromatography-tandem mass spectrometry(LC-MS/MS) identified different N-glycosylation sites in GP64(N94,N252,N266,N302,N348,N367,and N471) and SPΔnGP64(N302 and N471),none matching predicted *** showed that different positions affected fusion activity or infectivity to different extents,while all mutations were noninfectious in SPΔnGP64 recombinant *** conclusion,N-glycosylation of BmNPV GP64 follows a nonclassical pattern,and the retention of signal peptide significantly affects the glycosylation modification sites,which provides important information to elucidate the expression and secretion of GP

关键词： Baculovirus N-glycosylation signal peptide GP64 Viral infection embrane fusion

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A Novel signal peptide is Involvement of the NdhB Subunit Secretory Pathway in Soybean Chloroplast

A Novel Signal Peptide is Involvement of the NdhB Subunit Se...

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中国植物生理学会第十次会员代表大会暨全国学术年会

作者： Yi He1, Zhengjian Zhou1, Xiaofeng Li1, Dean Jiang1* 1State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China

＜正＞The NdhB subunit encoded by ndhB of NDH complex in the thylakoid membranes, commonly consists of 510 amino acids in soybean chloroplast[1]. However, the new NdhB subunit with only 492 amino acids in S111-

关键词： NdhB subunit signal peptide secretory pathway

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A combined method based on wavelet transform for signal peptide Local Features Recognition

A combined method based on wavelet transform for signal Pept...

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第九届全国计算（机）化学学术会议

作者： Yizhou Li Cuisong Zhou Zhining Wen Fuyuan Tan Menglong Li College of Chemistry,Sichuan University,Sichuan,Chengdu,610064,

＜正＞signal peptides direct nascent proteins to certain organelles and are cleaved off after the proteins *** the rapid increase of sequence entries,it is highly desirable to develop automated algorithm to identify the signal peptide or predict its cleavage ***,many methods have been *** vast majority of existing predictive methods resorted to a fixed window pattern,*** example,weight-matrix,an early computational method for predicting cleavage sites,employed a so-called summation window of（-13,+2）to predict cleavage sites of both eukaryotic and prokaryotic.A predictor signalP was developed for both signal peptide and its cleavage site based on neural *** applied different window patterns to five datasets:（-15, 4-4）to human,（-17,+2）to eukaryote,（-11,+2）to Gram-negative bacteria,（-21,+2）to Gram-positive bacteria and（-15,+2）to *** prompts us to propose a model that is universal for all signal peptides. The properties of signal peptides are known to differ between various types of organisms, including the length and *** has been reported that the location of cleavage sites are related to several local featUres in signal *** this context,studies on these local features might be helpful for constructing such a model and,even,for the further researches on signal *** construct such a model,it shall firstly encounter the problem that signal peptides are various in different organism *** a solution to this,the architecture of the model is separated into two *** the first step,h-region is *** this,the last three characteristic amino acids（Here,we call it（-3,-1）segment）of each signal peptide is *** the submodel of detecting h-region,a strategy of two-reconstructed profiles with wavelet transform was *** is well performed and demonstrated not affected by the variety of h-regious. This combined model is convenient to different organism *** predicting h-re

关键词： signal peptide Cleavage Site Wavelet Transform

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Missense mutations in the signal peptide of the porcine GH gene affect cellular synthesis and secretion

Missense mutations in the signal peptide of the porcine GH g...

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第七届中国畜牧科技论坛

作者： Cheng yunyun Liu songcai Lu chao Wu qingyan Li siming Fu haoyu Wang gang Lv chen Nie linyan Zhang yu Yu hao Hao linlin College of Animal Science Jilin University Five-Star Animal Health Pharmaceutical Factory of Jilin Province

In previous investigations,we have demonstrated the mutations in the signal peptide of porcine GH gene were associated with the *** this study,the fusion gene expression vectors which consisted of eight signal peptide mutants of GH gene and EGFP gene were constructed according to three missense mutations(***9 Ala,***22 Arg and ***25Gly),and they were transfected into the GH3 cell *** inhibition levels of EGFP gene transcriptions with different signal peptide mutants were significantly ***,the allelic variants carrying Val in codon 9 showed higher protein synthesis(Psignal sequence influence the expression and the secretion of the *** the best of our knowledge,this is the first report addressing the functional consequences of the mutations in the signal peptide of porcine GH gene.

关键词： GH gene signal peptide cellular secretion RNA fold

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