摘要
胶原蛋白独特的三股螺旋结构,使其具有良好的力学性能和生物相容性,在生物医学材料等方面有广泛应用。利用微生物表达重组胶原蛋白,具有发酵周期短、成本低、便于遗传操作等优点,近些年发展迅速,但分泌带有三股螺旋结构的重组胶原蛋白仍是研究的难点。该研究以来源于化脓性链球菌的胶原蛋白V-B为对象,搭建了谷氨酸棒杆菌分泌表达重组胶原蛋白的平台。在重组胶原蛋白V-B前端引入6种不同信号肽(CspB、PorB、Cg1514、CgR0949、Cg2052或TorA),构建重组表达载体,并转入食品级表达宿主谷氨酸棒状杆菌Corynebacterium glutamicum ATCC 13032中,结果显示PorB信号肽介导的分泌效果最好。此外,对PorB信号肽介导的分泌表达进行培养基、起始诱导菌浓度、诱导时长、IPTG浓度、溶氧的优化,最终胶原域B在摇瓶水平产量达到30 mg/L,是优化前的3倍,并通过圆二色谱表征,验证其正确折叠为三股螺旋。该研究为重组胶原蛋白在谷氨酸棒杆菌中的高效分泌表达奠定了基础。
Collagen is the most abundant structural protein in human body and a considerable component of cytoplasmic matrix.Collagen,attributed to its unique triple helix structure,has good mechanical properties and high biocompatibility,thereby being widely used in many applications,such as biomedicine,tissue engineering and cosmetics.The use of microorganisms to express recombinant collagen has the advantages of short fermentation cycle,low cost,and convenient genetic manipulation,which has been developed rapidly in recent years.However,the secretion of recombinant collagen with a triple helix structure remains a challenge.In this study,a platform for the secretion of recombinant collagen by Corynebacterium glutamicum was established.Six different signal peptides(Cg1514,Cg2052,CgR0949,CspB,PorB or TorA)were fused to the N-terminal of V-B derived from Streptococcus pyogenes and ligated into pXMJ19 to construct a recombinant expression vector.The recombinant plasmid was transferred into the food-grade expression host C.glutamicum ATCC 13032 to construct six recombinant strains.The secretion efficiency of these signal peptides was compared,and the expression conditions were optimized.The results showed that PorB was the most efficient signal peptide for V-B secretion.At the same time,the yields of collagen mediated by the Sec-dependent signal peptides Cg1514,CspB and PorB were higher than those of CgR0949 and TorA,which were the Tat-dependent signal peptides.This is consistent with the latest research result that C.glutamicum mainly secretes spider silk protein in a Sec-dependent manner.Therefore,it is speculated that the Sec-dependent secretion pathway is conducive to the secretion of fibrous protein such as collagen and spider silk protein.In addition,the culture medium,initial induction OD_(600),induction duration,IPTG concentration,and dissolved oxygen were optimized for the secretory expression mediated by PorB signal peptide.The optimal expression conditions were as follows:cultured in 500 mL baffled shake flask with 50 mL culture medium 1(20 g/L glucose,20 g/L corn steep powder,20 g/L(NH_(4))_(2) SO_(4),1 g/L KH_(2) PO_(4),1 g/L K_(2) HPO_(4),0.25 g/L MgSO_(4),and 42 g/L MOPS[3-(N-Morpholinyl)propanesulfonic acid sodium salt]),with an initial induction OD_(600) of 6 and induced by 0.5 mmol/L IPTG for 40 h.It was found that dissolved oxygen was the key factor affecting collagen production at the flask level,which provided guidance for further scale-up test at industrial production level.Subsequently,the supernatant was taken for affinity purification,followed by trypsin digestion.The V domain was cut into small peptides,while the collagen domain B was not digested due to its rigid triple helix structure when it’s correctly folded.After desalting,lyophilization and weighing,the final secretion yield of collagen domain B at the shake flask level reached 30 mg/L,which was 3 times that before optimization.The secondary structure was characterized by circular dichroism showing that it was correctly folded into a typical triple helix structure of collagen.The melting curve was scanned at 225 nm from 10℃to 70℃and the melting temperature(Tm)value of collagen domain B was calculated to be 25.82℃,which was close to that reported previously.The Corynebacterium glutamicum expression and secretion system established in this study is of food safety grade,and can efficiently secrete collagen with a triple helix structure,which provides a new host choice for the recombinant expression of collagen.Further,this development can provide solutions and theoretical basis for the production of recombinant collagen as well as other functional biomaterial proteins for the application in biomedicine and tissue engineering.
作者
齐静静
范炳森
张萌
许菲
QI Jingjing;FAN Bingsen;ZHANG Meng;XU Fei(Key Lab of Ministry of Education of Industrial Biotechnology,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2022年第15期9-17,共9页
Food and Fermentation Industries
基金
国家自然科学基金项目(22078129)
中央高校基本科研业务费专项资金(JUSRP121014)。
关键词
重组胶原蛋白
谷氨酸棒杆菌
信号肽
分泌表达
发酵条件优化
recombinant collagen
Corynebacterium glutamicum
signal peptide
secretion
expression optimization
