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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (11): 82-89    DOI: 10.13523/j.cb.2007046
综述     
重组贻贝足蛋白的研究进展与应用*
薛瑞,姚林,王瑞,罗正山,徐虹,李莎()
南京工业大学食品与轻工学院 材料化学工程国家重点实验室 南京 211800
Advances and Applications of Recombinant Mussel Foot Proteins
XUE Rui,YAO Lin,WANG Rui,LUO Zheng-shan,XU Hong,LI Sha()
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Food Science and Light Industry, Nanjing Tech University, Nanjing 211800, China
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摘要：

贻贝足蛋白是一类通过贻贝足腺分泌的蛋白质复合物,与基质表面发生反应而产生极强的黏附作用。其在海洋环境中具有强黏附能力、可降解性和优秀的生物相容性等优点,因此常被用做生物医药黏合剂。但提取天然蛋白质受原料来源限制,且工艺烦琐导致价格高昂,阻碍了贻贝足蛋白的进一步应用发展。微生物合成的最新进展为贻贝足蛋白的产出提供了一种新思路,并且具有扩大规模生产的意义。主要综述了贻贝足蛋白的基因工程生产方法,总结了重组蛋白在黏附抗污涂层、组织工程材料等领域的应用现状。同时对其研究方向进行了展望,指出重组贻贝足蛋白的进一步发展的关键技术是解析蛋白质的构效和层级结构,在此基础上提高其异源表达水平,以获得更多生物功效性的衍生产品。
关键词： 贻贝蛋白;  重组表达;  生物黏合剂    
Abstract:

Mussel foot proteins are a kind of protein complex secreted by mussel foot gland, which have strong adhesion to the surface of substrates due to reactions. Because of its strong adhesion, biodegradability and excellent biocompatibility in the marine environments, it is often used as bio-medical adhesives. However, the extraction of natural proteins is limited by the source of raw materials, and the complex process leads to high price, which hinders the further application and development of mussel foot proteins. The latest development of microbial synthesis provides a new way of thinking for mussel foot protein production and has the significance of expanding the scale of production. The production methods of mussel foot protein by genetic engineering were reviewed, and the applications of recombinant proteins in adhesion antifouling coatings and tissue engineering materials were summarized. At the same time, the research direction is prospected, and it is pointed out that the key technology for the further development of recombinant mussel foot proteins is to analyze the structure activity and hierarchical structure of the protein, on the basis of which the expression level of the protein is improved, so as to obtain more derivatives with biological efficacy.
Key words: Mussel proteins    Recombinant expression    Biological adhesives
收稿日期: 2020-07-26 出版日期: 2020-12-11
ZTFLH:  Q78  
基金资助: * 国家重点研发计划(2019YFA0905200);江苏省先进生物制造创新中心项目(XTB1804);材料化学工程国家重点实验室开放课题(KL19-12)
通讯作者: 李莎     E-mail: lisha@njtech.edu.cn
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薛瑞
姚林
王瑞
罗正山
徐虹
李莎

引用本文:

薛瑞,姚林,王瑞,罗正山,徐虹,李莎. 重组贻贝足蛋白的研究进展与应用*[J]. 中国生物工程杂志, 2020, 40(11): 82-89.

XUE Rui,YAO Lin,WANG Rui,LUO Zheng-shan,XU Hong,LI Sha. Advances and Applications of Recombinant Mussel Foot Proteins. China Biotechnology, 2020, 40(11): 82-89.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2007046    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I11/82

图1  不同贻贝蛋白在足丝中的分布
Protein type Molecular weight(kDa) Isoelectric point(pI) DOPA(mol%) Distribution
Mfp-1 ≈110 10.5 10-15 The cuticle layer of the byssus
Mfp-2 42-47 9-10 2-3 Plaque foam
Mfp-3 5-7 >10.5 20-25 Primer layer
Mfp-4 93 9 2 Thread collagen anchor
Mfp-5 9.5 9.5 25-30 Primer layer
Mfp-6 11.6 9.5 <5 Primer layer
表1  不同类型贻贝足丝蛋白的性质比较
Host Origin Protein
type		 Expressed fragment Molecular
weight
(kDa)		 Expression Reference
Escherichia
coli		 Mytilus
edulis		 Mefp-1 6 decapeptide repeats 7 Inclusion body [8]
7 decapeptide repeats; fused with OmpA signal peptide at N-terminus 8 The soluble expression was twice higher than without OmpA [18]
Mytilus
galloprovinciali		 Mgfp-5 Full-length 13.5 ≈ 40% of total cell proteins; mainly soluble [16]
Mgfp-3A Full-length 7 ≈56% of total cell proteins; half soluble [17]
In vivo DOPA incorporation by TyrRS 7 — [23]
fp-151 6 Mfp-1 decapeptide repeats at each Mfp-5 terminus 24.5 ≈40% of total cell proteins; inclusion body [20]
Co-expression with tyrosinase 24.5 Mainly soluble [22]
fp-353 Mfp-3A at each Mfp-5 terminus 22 ≈21% of total cell proteins; inclusion body [21]
Mytilus
coruscus		 Mcofp-1 12 decapeptide repeats 40.5 Soluble [11]
Mcofp-3 Full-length 9.18 Inclusion body [12]
Perna viridis Pvfp-1
(R-240)		 8 decapeptide repeats 26.78 Soluble [13]
Pvfp-1
(C-237)		 C-terminus non repetitive area 25.58 Soluble [13]
Yeast Mytilus
californianus		 Mcfp-3 Fused with first 9 amino acids of HA0 at N-terminus 7 Soluble [26]
Insect Sf9
cells		 Mytilus
galloprovinciali		 fp-151 6 Mfp-1 decapeptide repeats at each Mfp-5 terminus 24.5 ≈50% of total cell proteins; soluble [27]
Chicory Mytilus
galloprovinciali		 Mgfp-5 Full-length — The expression rate in T1 generation was 58.70% [29]
表2  基因工程法生产贻贝足蛋白的研究进展
[1] Harrington M J, Masic A, Holten-Andersen N, et al. Iron-clad fibers: a metal-based biological strategy for hard flexible coatings. Science, 2010,328(5975):216-220.
doi: 10.1126/science.1181044 pmid: 20203014
[2] Petrone L, Kumar A, Sutanto C N, et al. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins. Nature Communications, 2015,6(1):8737.
doi: 10.1038/ncomms9737
[3] Hwang D S, Waite J H. Three intrinsically unstructured mussel adhesive proteins, mfp-1, mfp-2, and mfp-3: analysis by circular dichroism. Protein Science, 2012,21(11):1689-1695.
doi: 10.1002/pro.2147
[4] Holten-Andersen N, Waite J H. Mussel-designed protective coatings for compliant substrates. Journal of Dental Research, 2008,87(8):701-709.
doi: 10.1177/154405910808700808 pmid: 18650539
[5] Kan Y, Danner E W, Israelachvili J N, et al. Boronate complex formation with Dopa containing mussel adhesive protein retards ph-induced oxidation and enables adhesion to mica. PLoS One, 2014,9(10):e108869.
pmid: 25303409
[6] Yu J, Wei W, Danner E, et al. Mussel protein adhesion depends on interprotein thiol-mediated redox modulation. Nature Chemical Biology, 2011,7(9):588-590.
doi: 10.1038/nchembio.630 pmid: 21804534
[7] Shin M, Shin J Y, Kim K, et al. The position of lysine controls the catechol-mediated surface adhesion and cohesion in underwater mussel adhesion. Journal of Colloid and Interface Science, 2020,563:168-176.
doi: 10.1016/j.jcis.2019.12.082 pmid: 31874305
[8] Kitamura M, Kawakami K, Nakamura N, et al. Expression of a model peptide of a marine mussel adhesive protein in Escherichia coli and characterization of its structural and functional properties. Journal of Polymer Science Part A: Polymer Chemistry, 1999,37(6):729-736.
[9] Hwang D S, Yoo H J, Jun J H, et al. Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli. Appl Environ Microbiol, 2004,70(6):3352-3359.
doi: 10.1128/AEM.70.6.3352-3359.2004 pmid: 15184131
[10] Hwang D S, Gim Y, Cha H J. Expression of functional recombinant mussel adhesive protein type 3A in Escherichia coli. Biotechnology Progress, 2005,21(3):965-970.
doi: 10.1021/bp050014e pmid: 15932281
[11] 蒋臻, 刘加鹏, 杨丙晔, 等. 厚壳贻贝粘附蛋白十肽重复序列的表达及功能分析. 中国生物化学与分子生物学报, 2010,26(3):275-282.
Jiang Z, Liu J P, Yang B Y, et al. Expression and functional analysis of deceptide repeat sequence of adhesive protein in Mytilus coruscus. Chinese Journal of Biochemistry and Molecular Biology, 2010,26(3):275-282.
[12] 李楠楠, 谭亮, 王智平, 等. 厚壳贻贝足丝黏附蛋白 mfp-3 重组表达及黏附功能分析. 中国生物化学与分子生物学报, 2011,27(9):851-857.
Li N N, Tan L, Wang Z P, et al. Recombinant expression and adhesion function analysis of thick-shell mussel foot silk adhesion protein mfp-3. Chinese Journal of Biochemistry and Molecular Biology, 2011,27(9):851-857.
[13] Jiang Z, Du LN, Ding X Y, et al. A novel recombinant bioadhesive designed from the non-repeating region of Perna viridis foot protein-1. Materials Science and Engineering: C, 2012,32(5):1280-1287.
[14] Guerette P A, Hoon S, Seow Y, et al. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nat Biotechnol, 2013,31(10):908-915.
doi: 10.1038/nbt.2671 pmid: 24013196
[15] Santonocito R, Venturella F, Dal Piaz F, et al. Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive. J Biol Chem, 2019; 294(34):12826-12835. DOI: 10.1074/jbc.RA119.009531.
doi: 10.1074/jbc.RA119.009531 pmid: 31292195
[16] Choi Y S, Kang D G, Lim S, et al. Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material. Biofouling, 2011,27(7):729-737.
doi: 10.1080/08927014.2011.600830 pmid: 21770718
[17] Yang B, Kang D G, Seo J H, et al. A comparative study on the bulk adhesive strength of the recombinant mussel adhesive protein fp-3. Biofouling, 2013,29(5):483-490.
doi: 10.1080/08927014.2013.782541 pmid: 23668263
[18] Lee S J, Han Y H, Nam B H, et al. A novel expression system for recombinant marine mussel adhesive protein Mefp1 using a truncated OmpA signal peptide. Molecules & Cells, 2008,26(1):34-40.
pmid: 18511886
[19] 吕玉伟, 吕亚维, 杨泽熵, 等. 贻贝蛋白 Mgfp-5 的原核表达与纯化. 西北大学学报(自然科学版), 2016, (3):16.
Lv Y W, Lv Y W, Yang Z S, et al. Prokaryotic expression and purification of mussel protein Mgfp-5. Journal of Northwest University (Natural Science Edition), 2016, (3):16.
[20] Hwang D S, Gim Y, Yoo H J, et al. Practical recombinant hybrid mussel bioadhesive fp-151. Biomaterials, 2007,28(24):3560-3568.
doi: 10.1016/j.biomaterials.2007.04.039 pmid: 17507090
[21] Gim Y, Hwang D S, Lim S, et al. Production of fusion mussel adhesive fp‐353 in Escherichia coli. Biotechnology Progress, 2008,24(6):1272-1277.
doi: 10.1002/btpr.65 pmid: 19194941
[22] Choi Y S, Yang Y J, Yang B, et al. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli. Microbial Cell Factories, 2012,11(1):139.
[23] Yang B, Ayyadural N, Yun H, et al. In vivo residue-specific Dopa-incorporated engineered mussel bioglue with enhanced adhesion and water resistance. Angewandte Chemie International Edition, 2014,53(49):13360-13364.
doi: 10.1002/anie.201406099 pmid: 25195781
[24] Jeong Y S, Yang B, Yang B, et al. Enhanced production of Dopa-incorporated mussel adhesive protein using engineered translational machineries. Biotechnology and Bioengineering, 2020,117(7):1961-1969.
doi: 10.1002/bit.27339 pmid: 32196642
[25] 李楠楠, 王智平, 鲁涛, 等. 厚壳贻贝足丝盘黏附蛋白 mcofp-3 的重组真核表达. 生物技术通报, 2010,1(12):148-153.
Li N N, Wang Z P, Lu T, et al. Recombinant eukaryotic expression of thick-shelled mussel foot silk disc adhesion protein mcofp-3. Biotechnology Bulletin, 2010,1(12):148-153.
[26] Platko J D, Deeg M, Thompson V, et al. Heterologous expression of Mytilus californianus foot protein three (Mcfp-3) in Kluyveromyces lactis. Protein Expression & Purification, 2008,57(1):0-62.
[27] Lim S, Kim K R, Choi Y S, et al. In vivo post‐translational modifications of recombinant mussel adhesive protein in insect cells. Biotechnology Progress, 2011,27(5):1390-1396.
doi: 10.1002/btpr.662 pmid: 21732552
[28] 吕玉伟, 张雨靖, 吕亚维, 等. 贻贝粘蛋白Mgfp-5基因在烟草中的转化. 基因组学与应用生物学, 2016,35(1):180-190.
Lv Y W, Zhang Y J, Lv Y W, et al. Transformation of mussel mucin Mgfp-5 gene in tobacco. Genomics and Applied Biology, 2016,35(1):180-190.
[29] 吕亚维. 贻贝粘合蛋白 Mgfp-5 基因在菊苣中的表达研究. 西安:西北大学, 2018.
Lv Y W. Study on the expression of mussel adhesion protein Mgfp-5 gene in chicory. Xi’an:Northwest University, 2018.
[30] Kim H J, Hwang B H, Lim S, et al. Mussel adhesion-employed water-immiscible fluid bioadhesive for urinary fistula sealing. Biomaterials, 2015,72:104-111.
doi: 10.1016/j.biomaterials.2015.08.055 pmid: 26352517
[31] Jo Y K, Seo J H, Choi B H, et al. Surface-independent antibacterial coating using silver nanoparticle-generating engineered mussel glue. ACS Applied Materials & Interfaces, 2014,6(22):20242-20253.
doi: 10.1021/am505784k pmid: 25311392
[32] Zhong C, Gurry T, Cheng A A, et al. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature Nanotechnology, 2014,9(10):858-866.
doi: 10.1038/nnano.2014.199 pmid: 25240674
[33] Zhang C, Huang J F, Zhang J C, et al. Engineered Bacillus subtilis biofilms as living glues. Materials Today, 2019,4(28):40-48.
[34] Qi H S, Zheng W W, Zhou X, et al. A mussel-inspired chimeric protein as a novel facile antifouling coating. Chemical Communications, 2018,54(80):11328-11331.
doi: 10.1039/c8cc05298k pmid: 30239536
[35] Qi H S, Zheng W W, Zhang C, et al. Novel mussel-inspired universal surface functionalization strategy: Protein-based coating with residue-specific post-translational modification in vivo. ACS Applied Materials & Interfaces, 2019,11(13):12846-12853.
[36] Qi H S, Zhang C, Guo H S, et al. Bioinspired multifunctional protein coating for antifogging, self-cleaning, and antimicrobial properties. ACS Applied Materials & Interfaces, 2019,11(27):24504-24511.
doi: 10.1021/acsami.9b03522 pmid: 31257848
[37] Jeon E Y, Hwang B H, Yang Y J, et al. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking. Biomaterials, 2015,67:11-19.
doi: 10.1016/j.biomaterials.2015.07.014 pmid: 26197411
[38] Hwang D S, Waite J H, Tirrell M. Promotion of osteoblast proliferation on complex coacervation-based hyaluronic acid-recombinant mussel adhesive protein coatings on titanium. Biomaterials, 2010,31(6):1080-1084.
doi: 10.1016/j.biomaterials.2009.10.041 pmid: 19892396
[39] Jo Y K, Choi B H, Kim C S, et al. Diatom‐inspired silica nanostructure coatings with controllable microroughness using an engineered mussel protein glue to accelerate bone growth on titanium‐based implants. Advanced Materials, 2017,29(46):1704906.
[40] Hwang D S, Sim S B, Cha H J. Cell adhesion biomaterial based on mussel adhesive protein fused with RGD peptide. Biomaterials, 2007,28(28):4039-4046.
doi: 10.1016/j.biomaterials.2007.05.028 pmid: 17574667
[41] Choi B H, Choi Y S, Kang D G, et al. Cell behavior on extracellular matrix mimic materials based on mussel adhesive protein fused with functional peptides. Biomaterials, 2010,31(34):8980-8988.
doi: 10.1016/j.biomaterials.2010.08.027 pmid: 20832110
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