Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selec...Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.展开更多
Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication syste...Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.展开更多
目的可致肿瘤细胞死亡的人α乳清蛋白(human alpha-lactalbumin made lethal to tumor cells,HAMLET)是一种可导致多种癌细胞死亡的强效选择性肿瘤杀伤蛋白质-脂酸复合物。为了使HAMLET应用于肿瘤的治疗并对其杀伤机制进行探讨,我们需...目的可致肿瘤细胞死亡的人α乳清蛋白(human alpha-lactalbumin made lethal to tumor cells,HAMLET)是一种可导致多种癌细胞死亡的强效选择性肿瘤杀伤蛋白质-脂酸复合物。为了使HAMLET应用于肿瘤的治疗并对其杀伤机制进行探讨,我们需要建立和优化简便、快速、规模化制备与纯化HAMLET的方法。方法通过构建人α乳清蛋白分泌型细胞MCF-7的cDNA文库,PCR扩增出编码人α乳清蛋白成熟肽段的全长序列,然后克隆入pET30a(+)表达载体质粒,并转化BL21(DE3)宿主大肠杆菌。经过IPTG诱导,表达细菌的裂解,蛋白体外复性,Ni-NTA亲和层析柱纯化等步骤,获得了高纯度的重组人α乳清蛋白的C端His标签融合蛋白。纯化蛋白经EDTA脱钙后,与油酸(oleic acid,OA)在加热条件下制备HAMLET复合物。同时,对HAMLET的理化特性和生物学活性进行检测。结果通过与经典方法制备的牛α乳清蛋白和OA复合物(BAMLET)进行比较,发现我们制备的HAMLET在紫外吸收光谱、疏水性质特征及肿瘤杀伤活力方面与BAMLET活性相符,并且可引起HeLa肿瘤细胞的凋亡样程序性死亡。结论本研究自主制备的HAMLET成本经济、方法简便且具有较好的规模化放大前景,为日后临床应用奠定基础。展开更多
基金This work was funded by National Natural Science Foundation of China(32272279)the Key R&D project of Qingdao Science and Technology Plan(22-3-3-hygg-29-hy).
文摘Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.
基金financially supported by the National Science Fund for Excellent Young Scholars(32222069)National Natural Science Foundation of China(32172349)+1 种基金Foundation for Innovative Research Groups of the National Natural Science Foundation of China(32021005)National Key Research and Development Program of China(2020YFA0908300).
文摘Bacillus thuringiensis,a safe bacterium widely used in agriculture for the biocontrol of pests,has great potential for protein production.The linear plasmid expression system,bacterial orthogonal DNA replication system constructed based on B.thuringiensis prophage GIL16,can achieve stable and high levels of gene expression in the absence of external selection pressure,facilitating development of B.thuringiensis chassis cells.However,the regulatory elements of gene expression and protein secretion suitable for the B.thuringiensis expression system are still lacking.Therefore,the development and optimization of different genetic tools are required.We constructed a promoter library containing 107 different-strength promoters(covering persistently high/intermediate/low level)by transcriptomic analysis of the cell at different growth stages and a signal peptide library(59 signal peptides from Bacillus subtilis and four endogenous signal peptides from B.thuringiensis)to enrich the genetic toolbox using alpha-lactalbumin(α-LA)as the characterization product.Then,a high-throughput microfluidic screening platform based on BacORep and self-assembled split fluorescent protein was developed to further optimize expression elements,resulting in an improved α-LA-producing B.thuringiensis.Finally,the maximum copy number of linear plasmids was 9.3 times higher than that of the original.The titer of α-LA reached 107.7 mg/L in a 3 L bioreactor,which was comparable to the highest yield reported in Komagataella phaffii.We substantially expanded the synthetic biology toolbox for linear plasmid expression systems and provided a strategy for creating efficient prokaryotic expression system.
文摘目的可致肿瘤细胞死亡的人α乳清蛋白(human alpha-lactalbumin made lethal to tumor cells,HAMLET)是一种可导致多种癌细胞死亡的强效选择性肿瘤杀伤蛋白质-脂酸复合物。为了使HAMLET应用于肿瘤的治疗并对其杀伤机制进行探讨,我们需要建立和优化简便、快速、规模化制备与纯化HAMLET的方法。方法通过构建人α乳清蛋白分泌型细胞MCF-7的cDNA文库,PCR扩增出编码人α乳清蛋白成熟肽段的全长序列,然后克隆入pET30a(+)表达载体质粒,并转化BL21(DE3)宿主大肠杆菌。经过IPTG诱导,表达细菌的裂解,蛋白体外复性,Ni-NTA亲和层析柱纯化等步骤,获得了高纯度的重组人α乳清蛋白的C端His标签融合蛋白。纯化蛋白经EDTA脱钙后,与油酸(oleic acid,OA)在加热条件下制备HAMLET复合物。同时,对HAMLET的理化特性和生物学活性进行检测。结果通过与经典方法制备的牛α乳清蛋白和OA复合物(BAMLET)进行比较,发现我们制备的HAMLET在紫外吸收光谱、疏水性质特征及肿瘤杀伤活力方面与BAMLET活性相符,并且可引起HeLa肿瘤细胞的凋亡样程序性死亡。结论本研究自主制备的HAMLET成本经济、方法简便且具有较好的规模化放大前景,为日后临床应用奠定基础。
基金the scientific and technological program of Fujian Science and Technology Department(2004N026)the Natural Science Foundation of Fujian Province(B0410009)the scientific and technological program of undergraduate students in Fujian Normal university(BKL2007062)
