毕赤酵母表达重组羟基化人源明胶的研究
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摘要
明胶是胶原的热变性和不可逆的化学降解产物,按用途不同可分为照相明胶、药用明胶、食用明胶和工业明胶,作为配料剂、悬浮剂、粘合剂、成型剂等广泛应用于各领域。传统意义上的商品化明胶作为一种屠宰场的副产品,主要通过物理化学方法提取,源于各种品种、来源和年龄的动物(每一批都不同),经过前处理后,明胶在-系列条件下序列是变化的。这样不同生产批次的明胶不相同并且具有不同的特性,再加上提取出来的明胶具有病毒和朊病毒的潜在污染的风险,难以完全保证其产品安全性,而重组DNA是获得明胶的另一种策略,这种策略通过表达具有特定长度和组成的胶原基因片段直接产生明胶,能够提供具有稳定的和可预测的特性以及具有确切分子组成的明胶。目前重组明胶由于没有引入外源脯氨酸羟化酶基因,因此都未能发生羟化,即使有的物种具有内源羟化能力,但羟化程度较低,从而不能形成坚固的三螺旋结构,在细胞内很容易降解,以至于不能较快地分泌到细胞外。
本论文通过引入外源脯氨酸羟化酶基因,构建多基因(明胶基因、脯氨酸羟化酶基因)共表达载体实现了在毕赤酵母中重组羟基化明胶的表达,并且获得了一定羟基化比例的明胶。在构建的载体中,三个独立
表达盒串联在一起,每一个表达盒都有自己的启动子和终止子,分别转录出各自的mRNA并翻译出不同的蛋白,同时在每一个表达盒中,我们选用了毕赤酵母的两种强诱导型启动子(PAOX1和PFLD1)分别作为这些基因的启动子元件。主要工作有以下几个方面：
1.通过重叠延伸PCR方法合成了一段长约300bp的编码100个氨基酸(第531-630)的人Ⅰ型胶原蛋白alpha(1)链明胶基因(Genebank登录号：P02452)；
2.成功克隆了构成表达盒中的各个元件,其中PFLD1启动子元件是以毕赤酵母基因组为模板扩增获得,同时用绿色荧光蛋白的表达检测了启动子的功能,αMF信号肽和TT(终止子)元件都是以表达载体pPIC9K直接作为模板分别扩增获得,脯氨酸羟化酶基因(αP4H和β)分别是以含有各自编码基因的cDNA克隆为模板扩增获得；
3.重点构建了多基因表达载体,将明胶基因以及脯氨酸羟化酶基因(αP4H和p)同时引入到同一个毕赤酵母表达载体pPIC9K上。首先获得含有各元件的T-载体,然后通过酶切连接方法将相邻的两个含有元件的T-载体依次连接组装在同一个pGEM-T载体上,最后将整个表达框从T载体上切下后移入到表达载体pPIC9K上；
4.表达载体用SalⅠ线性化后,经电转化通过基因同源交换,整个表达框被整合到毕赤酵母KM71染色体中,长出的转化子都是甲醇利用慢型,用含有4mg/L G418的MD平板筛选后获得高拷贝菌株,经过甲醇诱导后,目的蛋白具有较强的诱导表达模式,分子量是单体(未羟化的明胶)的三倍左右,蛋白经LC-MS/MS检测与人胶原蛋白Ⅰ型相匹配,并且检测到在Gly-X-Y三联体的Y位置上大约有66%左右的脯氨酸发生了羟化作用；
5.对重组明胶的微生物生产进行了初步的发酵条件优化工作,诱导时间做到第六天时产量还有增长趋势,因此在诱导时间上可能还有进一步增产的空间,甲醇添加浓度以1%(每12小时一次)为最佳浓度。并且对表达的重组明胶进行了鉴定及表征工作。经圆二色谱检测,明胶的二级结构含有61.5%的β转角和38.5%的无规则卷曲,而不含传统的α螺旋和β折叠。明胶的核磁共振波谱检测结果表明：1H谱和13C谱都有与之相对应的特征性位移峰。明胶的紫外光谱显示它的最大吸收峰在225nm处。
Gelatin is a class of collagen thermal denaturation and irreversible chemical degradation products, and it can be categorized as photographic gelatin, medicine gelatin, edible gelatin, and industrial gelatin according to different uses, and used widely in various fields as an ingredient agent, suspending agent, adhesive agent and forming agent. As a slaughterhouse by-products, the commercialization gelatin in the traditional sense was mainly extracted by physical and chemical methods, therefore, different production batches of gelatin is generally not the same and have different characteristics, coupled with the extracted gelatin possesses the risk of potential contamination with viruses and prions, which make it difficult to fully guarantee their product safety, while recombinant DNA technology is another strategy to obtain gelatin.This strategy is produce gelatin directly by expressing collagen gene fragments with a specific length and composition, which can provide a gelatin with stable and predictable characteristics, and having the exact molecular composition. To date, all recombinant gelatins have no hydroxylation without the introduction of the exogenous prolyl 4-hydroxylase genes, and even some species have the endogenous hydroxylation capacity, but the level of hydroxylation was low, and thus can not form a solid three-helix structure, and it is easily degradable in cells that can not be rapidly secreted out of the cell.
In this thesis, expression of recombinant gelatin was achieved in Pichia pastoris, and a certain hydroxylation of gelatin was gain by introducing exogenous prolyl 4-hydroxylase genes. During the process of constructing the vector, three independent expression cassettes were connected one after another, and each expression cassette has its own promoter and terminator, respectively. In this way, their mRNAs were transcripted, respectively and translated into different proteins. In each expression cassette, we used two strong inducible promoter (PAOX1 and PFLD1), respectively as promoting elements of these genes. The main works are the following aspects:
1. Synthesized a length of about 300bp encoding 100 amino acids (No.531-630) of the human typeⅠcollagen alpha (1) gelatin chain gene (Genebank login:P02452) by overlap extension PCR method.
2. The components of expression cassette were successfully cloned. PFLD1 promoter was amplified with Pichia pastoris genomic DNA as a template, and the expression of green fluorescent protein was used to detect the function of the promoter; aMF signal peptide and TT (terminator) is amplified, separately with the expression vector pPIC9K as a template; prolyl 4-hydroxylase genes (αP4H andβ) was amplified, separately with the cDNA clones containing their coding gene as a template.
3. We constructed multi-gene expression vector with the gelatin gene and prolyl 4-hydroxylase gene (αP4H andβ) introducing into the same yeast expression vector pPIC9K. First, the components were cloned, then connect every two adjacent vectors containing the components by digestion and connection method in order to assemble the cloning vector onto the same pGEM-T vector, and finally the entire expression cassette was cut from the T-vector and inserted into the expression vector pPIC9K.
4. Expression vector was linearized with Sal I, and homologous exchange by electroporation, the entire expression cassette was integrated into the chromosome of Pichia pastoris KM71. All transformants were Muts, and MD plate containing 4mg/L G418 was used to screen high copy strains. After methanol induction, there is a protein band showing a strongly induced expression patterns. The molecular weight of the protein is about three times size of the monomer (non-hydroxylated gelatin). The protein was detected by LC-MS/MS, which indicated it matches with human type I collagen, and 66% hydroxylation of proline in the Y position of Gly-X-Y was occurred.
5. The fermentation conditions of microbial production of recombinant gelatin were preliminary optimized. The yield on the sixth day is still presented a growth trend-thus there is room for further increase in the yield of recombinant gelatin; The methanol concentration of 1% (once every 12 hours) is the most optimal concentration. The expressed recombinant gelatins were identified and characterizated. Circular dichroism detection indicated the secondary structure of gelatin contains 61.5%β-turn and 38.5% random coil without the traditional a-helix and P-sheet. The nuclear magnetic resonance spectroscopy of gelatin test results show that the 1H and 13C spectra have many corresponding characteristic displacement peaks. UV spectra of gelatin shows its maximum absorption peak is 225nm.
本论文通过引入外源脯氨酸羟化酶基因,构建多基因(明胶基因、脯氨酸羟化酶基因)共表达载体实现了在毕赤酵母中重组羟基化明胶的表达,并且获得了一定羟基化比例的明胶。在构建的载体中,三个独立
表达盒串联在一起,每一个表达盒都有自己的启动子和终止子,分别转录出各自的mRNA并翻译出不同的蛋白,同时在每一个表达盒中,我们选用了毕赤酵母的两种强诱导型启动子(PAOX1和PFLD1)分别作为这些基因的启动子元件。主要工作有以下几个方面：
1.通过重叠延伸PCR方法合成了一段长约300bp的编码100个氨基酸(第531-630)的人Ⅰ型胶原蛋白alpha(1)链明胶基因(Genebank登录号：P02452)；
2.成功克隆了构成表达盒中的各个元件,其中PFLD1启动子元件是以毕赤酵母基因组为模板扩增获得,同时用绿色荧光蛋白的表达检测了启动子的功能,αMF信号肽和TT(终止子)元件都是以表达载体pPIC9K直接作为模板分别扩增获得,脯氨酸羟化酶基因(αP4H和β)分别是以含有各自编码基因的cDNA克隆为模板扩增获得；
3.重点构建了多基因表达载体,将明胶基因以及脯氨酸羟化酶基因(αP4H和p)同时引入到同一个毕赤酵母表达载体pPIC9K上。首先获得含有各元件的T-载体,然后通过酶切连接方法将相邻的两个含有元件的T-载体依次连接组装在同一个pGEM-T载体上,最后将整个表达框从T载体上切下后移入到表达载体pPIC9K上；
4.表达载体用SalⅠ线性化后,经电转化通过基因同源交换,整个表达框被整合到毕赤酵母KM71染色体中,长出的转化子都是甲醇利用慢型,用含有4mg/L G418的MD平板筛选后获得高拷贝菌株,经过甲醇诱导后,目的蛋白具有较强的诱导表达模式,分子量是单体(未羟化的明胶)的三倍左右,蛋白经LC-MS/MS检测与人胶原蛋白Ⅰ型相匹配,并且检测到在Gly-X-Y三联体的Y位置上大约有66%左右的脯氨酸发生了羟化作用；
5.对重组明胶的微生物生产进行了初步的发酵条件优化工作,诱导时间做到第六天时产量还有增长趋势,因此在诱导时间上可能还有进一步增产的空间,甲醇添加浓度以1%(每12小时一次)为最佳浓度。并且对表达的重组明胶进行了鉴定及表征工作。经圆二色谱检测,明胶的二级结构含有61.5%的β转角和38.5%的无规则卷曲,而不含传统的α螺旋和β折叠。明胶的核磁共振波谱检测结果表明：1H谱和13C谱都有与之相对应的特征性位移峰。明胶的紫外光谱显示它的最大吸收峰在225nm处。
Gelatin is a class of collagen thermal denaturation and irreversible chemical degradation products, and it can be categorized as photographic gelatin, medicine gelatin, edible gelatin, and industrial gelatin according to different uses, and used widely in various fields as an ingredient agent, suspending agent, adhesive agent and forming agent. As a slaughterhouse by-products, the commercialization gelatin in the traditional sense was mainly extracted by physical and chemical methods, therefore, different production batches of gelatin is generally not the same and have different characteristics, coupled with the extracted gelatin possesses the risk of potential contamination with viruses and prions, which make it difficult to fully guarantee their product safety, while recombinant DNA technology is another strategy to obtain gelatin.This strategy is produce gelatin directly by expressing collagen gene fragments with a specific length and composition, which can provide a gelatin with stable and predictable characteristics, and having the exact molecular composition. To date, all recombinant gelatins have no hydroxylation without the introduction of the exogenous prolyl 4-hydroxylase genes, and even some species have the endogenous hydroxylation capacity, but the level of hydroxylation was low, and thus can not form a solid three-helix structure, and it is easily degradable in cells that can not be rapidly secreted out of the cell.
In this thesis, expression of recombinant gelatin was achieved in Pichia pastoris, and a certain hydroxylation of gelatin was gain by introducing exogenous prolyl 4-hydroxylase genes. During the process of constructing the vector, three independent expression cassettes were connected one after another, and each expression cassette has its own promoter and terminator, respectively. In this way, their mRNAs were transcripted, respectively and translated into different proteins. In each expression cassette, we used two strong inducible promoter (PAOX1 and PFLD1), respectively as promoting elements of these genes. The main works are the following aspects:
1. Synthesized a length of about 300bp encoding 100 amino acids (No.531-630) of the human typeⅠcollagen alpha (1) gelatin chain gene (Genebank login:P02452) by overlap extension PCR method.
2. The components of expression cassette were successfully cloned. PFLD1 promoter was amplified with Pichia pastoris genomic DNA as a template, and the expression of green fluorescent protein was used to detect the function of the promoter; aMF signal peptide and TT (terminator) is amplified, separately with the expression vector pPIC9K as a template; prolyl 4-hydroxylase genes (αP4H andβ) was amplified, separately with the cDNA clones containing their coding gene as a template.
3. We constructed multi-gene expression vector with the gelatin gene and prolyl 4-hydroxylase gene (αP4H andβ) introducing into the same yeast expression vector pPIC9K. First, the components were cloned, then connect every two adjacent vectors containing the components by digestion and connection method in order to assemble the cloning vector onto the same pGEM-T vector, and finally the entire expression cassette was cut from the T-vector and inserted into the expression vector pPIC9K.
4. Expression vector was linearized with Sal I, and homologous exchange by electroporation, the entire expression cassette was integrated into the chromosome of Pichia pastoris KM71. All transformants were Muts, and MD plate containing 4mg/L G418 was used to screen high copy strains. After methanol induction, there is a protein band showing a strongly induced expression patterns. The molecular weight of the protein is about three times size of the monomer (non-hydroxylated gelatin). The protein was detected by LC-MS/MS, which indicated it matches with human type I collagen, and 66% hydroxylation of proline in the Y position of Gly-X-Y was occurred.
5. The fermentation conditions of microbial production of recombinant gelatin were preliminary optimized. The yield on the sixth day is still presented a growth trend-thus there is room for further increase in the yield of recombinant gelatin; The methanol concentration of 1% (once every 12 hours) is the most optimal concentration. The expressed recombinant gelatins were identified and characterizated. Circular dichroism detection indicated the secondary structure of gelatin contains 61.5%β-turn and 38.5% random coil without the traditional a-helix and P-sheet. The nuclear magnetic resonance spectroscopy of gelatin test results show that the 1H and 13C spectra have many corresponding characteristic displacement peaks. UV spectra of gelatin shows its maximum absorption peak is 225nm.
引文
[1]彭必先,陈丽娟.从胶原到明胶[J].明胶科学与技术,1994,14(1)：1-11
[2]王师俊.明胶[J].化学世界,1991,32(2)：49-55
[3]王元荪.降低明胶灰分和重金属含量的方法[J].明胶科学与技术,2005,25(4)：186-188
[4]关林波,但卫华,曾睿.明胶及其在生物材料中的应用[J].材料导报,2006,20(11)：380-383
[5]徐润.明胶的生产及应用技术[M].北京：中国轻工业出版社,2000：1-270
[6]陈秀金,曹健美.胶原蛋白和明胶在食品中的应用[J].郑州工程学院学报,2002,23(1)：66-69
[7]史京京,王颖,王智慧,陈丽娟,彭必先.明胶组分含量与交联剂反应的研究[J].明胶科学与技术,2001,21(1)：23-30
[8]李卫林,曹健,汤克勇,左锦静,王岩.胶原蛋白结构和稳定性关系研究[J].中国皮革,2005,34(23)：14-16
[9]顾其胜,蒋丽霞.胶原蛋白与临床医学[M].上海：第二军医大学出版社,2003：1-58
[10]彭必先,陈丽娟.从胶原到明胶[J].明胶科学与技术,1994,14(2)：57
[11]安锋利,王建林,权美平,李璇.胶原蛋白的应用及发展前景[J].贵州农业科学,2011,39(1)：8-11
[12]Tuderman L, Myllyla R, Kivirikko KI. Mechanism of the Prolyl Hydroxylase Reaction[J]. Eur. J. Biochem.,1977,80:341-348
[13]Kivirikko KI, Pihlajaniemi T. Protein hydroxylation:prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit[J]. Faseb. J.,1989,3:1609-1617
[14]蒋挺大.胶原蛋白[M].北京：化学工业出版社,2001
[15]汪多仁.明胶[J].中国食品用化学品,1998,4：38-41
[16]位绍红,许永安.明胶提取工艺及其应用的研究进展[J].福建水产,2007,2：67-71
[17]王式箴译.明胶—全球的供应和需求[J].中国食品添加剂,1996,2：56-59
[18]Werten MWT, Wisselink WH. Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris[J]. Protein. Eng.,2001,14:447-454
[19]Werten MWT, van den Bosch TJ, Wind RD, Mooibroek H and de Wolf FA. High-yield secretion of recombinant gelatins by Pichia pastoris[J]. Yeast.,1999,15: 1087-1096
[20]de Bruin EC, de Wolf FA, Laane NCM. Expression and secretion of human α1(1)procollagen fragment by Hansenula polymorpha as compared to Pichia pastoris[J].Enzyme. Microb. Tech.,2000,26:640-644
[21]Zhang C, Baez J and Glatz CE. Purification and Characterization of a 44-kDa Recombinant Collagen Ⅰ α1 fragment from Corn Grain [J]. J. Agric. Food. Chem., 2009,57:880-887
[22]Olsen D, Yang C, Bodo M. Recombinant collagen and gelatin for drug delivery[J]. Adv. Drug. De.,2003,55:1547-1567
[23]Baez J, Olsen D, Polarek JW. Recombinant microbial systems for the production of human collagen and gelatin[J]. Appl. Microbiol. Biotechnol.,2005, 69:245-252
[24]Kivirikko KI, Myllyharju J. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit[J]. Matrix. Biol.,1998,16:357-368
[25]Kivirikko KI, Pihlajaniemi T. Collagen hydroxylases and the protein disulfide isomerase subunit of proly14-hydroxylases[J]. Adv. Enzymol. Relat. Aras. Mol.Biol.,1998,72:325-398
[26]Kuutti ER, Tuderman L,Kivirikko KL. Human prolyl hydroxylase[J]. Eur. J. Bioch.,1975,57:181-188
[27]Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis[J]. Matrix. Biol.,2003,22:15-24
[28]Vuorela A, Myllyharju J, Pihlajaniemi T. Coexpression with collagen markedly increases the half-life of the recombinant human prolyl 4-hydroxylase tetramer in the yeast Pichia pastoris[J]. Matrix. Biol.,1999,18:519-522
[29]Vuorela A, Myllyharju J, Nissi R, Pihlajaniemi T, Kivirikko KI. Assembly of human prolyl 4-hydroxylase and type Ⅲ collagen in the Pichia pastoris: formation of a stable enzyme tetramer requires co-expression with collagen and assembly of a stable collagen requires co-expression with prolyl 4-hydroxylase[J].Embo. J.,1997,16:6702-6712
[30]Gellissen G. Heterologous protein production in methylotrophic yeasts[J]. Appl. Microbiol. Biotechnol.,2000,54:741-750
[31]Schmidt FR. Recombinant expression systems in the pharmaceutical industry[J]. Appl. Microbiol. Biotechnol.,2004,65:363-372
[32]Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and product ion [J]. J. Mol. Recognit.,2005,18:119-138
[33]Ellis SB, Brust PF, Koutz PJ. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris[J]. Mol. Cell.Biol.,1985,5:1111-1121
[34]Shen S, Sulter G, Jeffries TW and Cregg JM. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris[J]. Gene.,1998,216:93-102
[35]Cregg JM, Cereghino JL. Recombinant protein expression in Pichia pastoris[J]. Mol. Biotechnol.,2000,16:23-52
[36]Cos O, Resina D, Ferrer P, Montesinos JL, Valero F. Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures[J]. Biochem. Eng. J.,2005,26:86-94
[37]赵翔,霍克克.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3)：308-311
[38]胡显文,高丽华.炭疸毒素受体ATR cDNA的合成和克隆及ATR-Fc融合蛋白的真核表达载体的构建[J].中国生物工程杂志,2005,25(12)：1-8
[39]Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension[J]. Gene.,1989,77:61-68
[40]何有节,张迈华,刘其则,王允肃,何先祺,朱正和.明胶的标准1H和13C—NMR谱[J].皮革科学与工程,1990,2：5-10
[41]Toman PD, Chisholm G, McMullin H. Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae[J]. J. Biol.Chem.,2000,275:23303-23309
[42]Bertrand E, Castanotto D. The expression cassette determines the functional activity of ribozymes in cells by controlling their intracellular localization[J]. RNA.,1997,3:75-88
[43]Kajino T, Takahashi H, Hirai M, and Yamada Y. Efficient production of artificially designed gelatins with a Bacillus brevis System[J]. Appl.Environ. Microb.,2000,66:304-309
[44]Asghar A, Henrickson R L. Chemical, biochemical, functional, and nutritional characteristics of collagen in food systems[J]. Adv. Food. Res.,1982,28: 231-372
[45]Kielty CM, Hopkinson I, Grant ME. The collagen family:structure, assembly and organization in the extracellular matrix. In:connective tissue and its heritable disorders[M]. Molecular, genetic and medical aspects Royce, P.M. and Steinmann, B., Eds.; Wiley-Liss:New York,1993
[46]Prockop DJ, Kivirikko KI. Collagens:molecular biology, diseases, and potentials for therapy[J]. Ann. Rev. Biochem.,1995,64:403-434
[47]Bateman JF, Lamande S, Ramshaw JAM. Collagen superfamily in extracellular matrix Molecular components and interactions[M]. WD Comper ed.; Harwood Academic Publishers Amsterdam.1995
[48]Kivirikko KI, Myllyla R, Pihlajaniemi T. Hydroxylation of proline and lysine residues in collagens and other animal and plant proteins[M]. In: Post-translational modifications of proteins, Harding, JJ. and Crabbe, MJC., Eds.; CRC Press, Boca Raton, FL.1992
[49]Olsen D, Jiang J, Chang R, Duffy R, Sakaguchi M, Leigh S, Lundgard R, Ju J, Buschman F, Truong-Le V, Pham B, Polarek JW. Expression and characterization of a low molecular weight recombinant human gelatin:development of a substitute for animal-derived gelatin with superior features[J]. Protein. Expr. Purif.,2005,40:346-357
[50]Chalfie M. Green fluorescent protein[J]. Photochem. Photobiol.,1995,62: 651-656
[51]Cereghino GPL, Cereghino JL. Non-repressing carbon sources for alcohol oxidase AOX1 promoter of Pichia pastoris[J]. Curr. Opin. Biotech.,2002,13:329-332
[52]Kivirikko KI, Helaakoski T, Tasanen K, Vuori K, Myllyla R, Parkkonen T, Pihlajaniemi T. Molecular biology of prolyl 4-hydroxylase[J]. Ann. N. Y. Acad. Sci.,1990,580:132-14
[53]Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation[J]. J. Biol. Chem.,1991,266:19867-19870
[54]宋小岩,黄雅钦,黄明智.明胶应用技术某些新进展[J].明胶科学与技术,2002,22(1)：1-6
[55]Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension[J]. Gene.,1989,77:61-68
[56]Warrens AN, Jones MD, Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification:an improved technique for the generation of hybrid proteins of immunological interest[J]. Gene.,1997,186:29-35
[57]萨姆布鲁克. 分子克隆实验指南[M].北京：科学出版社,1992
[58]陈红英,崔保安,李新生.融合基因mChIL-18/mChIFN-α克隆及其原核表达载体的构建[J].农业生物技术学报,2007,15(3)：388-392
[59]帅兴华,王少强,宋增峰,谢益民.明胶应用性能的研究[J].皮革化工,2007,24(3)：20-23
[60]Resina D, Cos O, Ferrer P, Valero F. Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter [J]. Biotechnol. Bioeng.,2005,91:760-767
[61]Pihlajaniemi T, Helaakoski T, Tasanen K, Myllyla R, Huhtala ML, Koivu J, Kivirikko K. Molecular cloning of the β-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene[J]. Embo. J.,1987,6:643-649
[62]Helaakoski T, Vuori K, Myllyla R, Kivirikko K, Pihlajaniemi T. Molecular cloning of the α-subunit of human prolyl 4-hydroxylase:The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts[J].Proc. Natl.Acad. Sci.,1989,86:4392-4396
[63]Bruin EC, Werten MWT, Laane C. Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin[J].Fems. Yeast. Res.,2002,1:291-298
[64]Vaughan PR, Galanis M, Richards KM, Tebb TA, Ramshaw JAM, Werkmeister JA. Production of Recombinant Hydroxylated Human Type Ⅲ Collagen Fragment in Saccharomyces cerevisiae[J]. DNA. Cell. Biol.,1998,17:511-518
[65]Kivirikko KI, Myllyharju J. Prolyl 4-hydroxylases and their protein disulf ide isomerase subunit[J]. Matrix.Biol.,1998,16:357-368
[66]Gelse K, Poschl E, Aigner T. Collagens-structure, function, and biosynthesis[J]. Adv. Drug. Deliver. Rev.,2003,55:1531-1546
[67]Bulleid NJ, John DCA, Kadler KE. Recombinant expression systems for the production of collagen[J]. Biochem. Soc. Trans.,2000,28:350
[68]Huang J, Foo CWP, Kaplan DL.Biosynthesis and application of silk-like and collagen-like proteins[J]. Polymer. Rev.,2007,47:29-62
[69]Myllyharju J, Nokelainen M, Vuorela A, Kivirikko KI. Expression of recombinant human type Ⅰ-Ⅲ collagens in the yeast Pichia pastoris[J]. Biochem. Soc. T.,2000,28:353-357
[70]Sreekrishna K, Brankamp RG, Kropp KE. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris[J]. Gene.,1997,190:55-62
[71]Wagner K, Poschl E, Turnay J, Balk JM. Coexpression of α and β subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen[J]. Biochem. J.,2000,352:907-911
[72]Gunnink IK, Vuorela A, Myllyharju J. Accumulation of properly folded human type Ⅲ procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris[J]. Matrix. Biol.,2000,19:29-36
[73]Pakkanen O, Pirskanen A, Myllyharju J. Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris[J]. J. Biotechnol.,2006,123:248-256
[74]Murray WD, Duff SJB, Lanthier PH. Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris[J]. Appl. Microbiol. Biotechnol.,1989,32:95-100
[75]王镜岩,朱圣庚,徐长法.生物化学[M].北京：高等教育出版社,2002,215
[76]Lin YK, Liu DC. Comparison of physical-chemical properties of type Ⅰ collagen from different species[J]. Food. Chem.,2006,99:244-251
[2]王师俊.明胶[J].化学世界,1991,32(2)：49-55
[3]王元荪.降低明胶灰分和重金属含量的方法[J].明胶科学与技术,2005,25(4)：186-188
[4]关林波,但卫华,曾睿.明胶及其在生物材料中的应用[J].材料导报,2006,20(11)：380-383
[5]徐润.明胶的生产及应用技术[M].北京：中国轻工业出版社,2000：1-270
[6]陈秀金,曹健美.胶原蛋白和明胶在食品中的应用[J].郑州工程学院学报,2002,23(1)：66-69
[7]史京京,王颖,王智慧,陈丽娟,彭必先.明胶组分含量与交联剂反应的研究[J].明胶科学与技术,2001,21(1)：23-30
[8]李卫林,曹健,汤克勇,左锦静,王岩.胶原蛋白结构和稳定性关系研究[J].中国皮革,2005,34(23)：14-16
[9]顾其胜,蒋丽霞.胶原蛋白与临床医学[M].上海：第二军医大学出版社,2003：1-58
[10]彭必先,陈丽娟.从胶原到明胶[J].明胶科学与技术,1994,14(2)：57
[11]安锋利,王建林,权美平,李璇.胶原蛋白的应用及发展前景[J].贵州农业科学,2011,39(1)：8-11
[12]Tuderman L, Myllyla R, Kivirikko KI. Mechanism of the Prolyl Hydroxylase Reaction[J]. Eur. J. Biochem.,1977,80:341-348
[13]Kivirikko KI, Pihlajaniemi T. Protein hydroxylation:prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit[J]. Faseb. J.,1989,3:1609-1617
[14]蒋挺大.胶原蛋白[M].北京：化学工业出版社,2001
[15]汪多仁.明胶[J].中国食品用化学品,1998,4：38-41
[16]位绍红,许永安.明胶提取工艺及其应用的研究进展[J].福建水产,2007,2：67-71
[17]王式箴译.明胶—全球的供应和需求[J].中国食品添加剂,1996,2：56-59
[18]Werten MWT, Wisselink WH. Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris[J]. Protein. Eng.,2001,14:447-454
[19]Werten MWT, van den Bosch TJ, Wind RD, Mooibroek H and de Wolf FA. High-yield secretion of recombinant gelatins by Pichia pastoris[J]. Yeast.,1999,15: 1087-1096
[20]de Bruin EC, de Wolf FA, Laane NCM. Expression and secretion of human α1(1)procollagen fragment by Hansenula polymorpha as compared to Pichia pastoris[J].Enzyme. Microb. Tech.,2000,26:640-644
[21]Zhang C, Baez J and Glatz CE. Purification and Characterization of a 44-kDa Recombinant Collagen Ⅰ α1 fragment from Corn Grain [J]. J. Agric. Food. Chem., 2009,57:880-887
[22]Olsen D, Yang C, Bodo M. Recombinant collagen and gelatin for drug delivery[J]. Adv. Drug. De.,2003,55:1547-1567
[23]Baez J, Olsen D, Polarek JW. Recombinant microbial systems for the production of human collagen and gelatin[J]. Appl. Microbiol. Biotechnol.,2005, 69:245-252
[24]Kivirikko KI, Myllyharju J. Prolyl 4-hydroxylases and their protein disulfide isomerase subunit[J]. Matrix. Biol.,1998,16:357-368
[25]Kivirikko KI, Pihlajaniemi T. Collagen hydroxylases and the protein disulfide isomerase subunit of proly14-hydroxylases[J]. Adv. Enzymol. Relat. Aras. Mol.Biol.,1998,72:325-398
[26]Kuutti ER, Tuderman L,Kivirikko KL. Human prolyl hydroxylase[J]. Eur. J. Bioch.,1975,57:181-188
[27]Myllyharju J. Prolyl 4-hydroxylases, the key enzymes of collagen biosynthesis[J]. Matrix. Biol.,2003,22:15-24
[28]Vuorela A, Myllyharju J, Pihlajaniemi T. Coexpression with collagen markedly increases the half-life of the recombinant human prolyl 4-hydroxylase tetramer in the yeast Pichia pastoris[J]. Matrix. Biol.,1999,18:519-522
[29]Vuorela A, Myllyharju J, Nissi R, Pihlajaniemi T, Kivirikko KI. Assembly of human prolyl 4-hydroxylase and type Ⅲ collagen in the Pichia pastoris: formation of a stable enzyme tetramer requires co-expression with collagen and assembly of a stable collagen requires co-expression with prolyl 4-hydroxylase[J].Embo. J.,1997,16:6702-6712
[30]Gellissen G. Heterologous protein production in methylotrophic yeasts[J]. Appl. Microbiol. Biotechnol.,2000,54:741-750
[31]Schmidt FR. Recombinant expression systems in the pharmaceutical industry[J]. Appl. Microbiol. Biotechnol.,2004,65:363-372
[32]Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and product ion [J]. J. Mol. Recognit.,2005,18:119-138
[33]Ellis SB, Brust PF, Koutz PJ. Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris[J]. Mol. Cell.Biol.,1985,5:1111-1121
[34]Shen S, Sulter G, Jeffries TW and Cregg JM. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris[J]. Gene.,1998,216:93-102
[35]Cregg JM, Cereghino JL. Recombinant protein expression in Pichia pastoris[J]. Mol. Biotechnol.,2000,16:23-52
[36]Cos O, Resina D, Ferrer P, Montesinos JL, Valero F. Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures[J]. Biochem. Eng. J.,2005,26:86-94
[37]赵翔,霍克克.毕赤酵母的密码子用法分析[J].生物工程学报,2000,16(3)：308-311
[38]胡显文,高丽华.炭疸毒素受体ATR cDNA的合成和克隆及ATR-Fc融合蛋白的真核表达载体的构建[J].中国生物工程杂志,2005,25(12)：1-8
[39]Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension[J]. Gene.,1989,77:61-68
[40]何有节,张迈华,刘其则,王允肃,何先祺,朱正和.明胶的标准1H和13C—NMR谱[J].皮革科学与工程,1990,2：5-10
[41]Toman PD, Chisholm G, McMullin H. Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae[J]. J. Biol.Chem.,2000,275:23303-23309
[42]Bertrand E, Castanotto D. The expression cassette determines the functional activity of ribozymes in cells by controlling their intracellular localization[J]. RNA.,1997,3:75-88
[43]Kajino T, Takahashi H, Hirai M, and Yamada Y. Efficient production of artificially designed gelatins with a Bacillus brevis System[J]. Appl.Environ. Microb.,2000,66:304-309
[44]Asghar A, Henrickson R L. Chemical, biochemical, functional, and nutritional characteristics of collagen in food systems[J]. Adv. Food. Res.,1982,28: 231-372
[45]Kielty CM, Hopkinson I, Grant ME. The collagen family:structure, assembly and organization in the extracellular matrix. In:connective tissue and its heritable disorders[M]. Molecular, genetic and medical aspects Royce, P.M. and Steinmann, B., Eds.; Wiley-Liss:New York,1993
[46]Prockop DJ, Kivirikko KI. Collagens:molecular biology, diseases, and potentials for therapy[J]. Ann. Rev. Biochem.,1995,64:403-434
[47]Bateman JF, Lamande S, Ramshaw JAM. Collagen superfamily in extracellular matrix Molecular components and interactions[M]. WD Comper ed.; Harwood Academic Publishers Amsterdam.1995
[48]Kivirikko KI, Myllyla R, Pihlajaniemi T. Hydroxylation of proline and lysine residues in collagens and other animal and plant proteins[M]. In: Post-translational modifications of proteins, Harding, JJ. and Crabbe, MJC., Eds.; CRC Press, Boca Raton, FL.1992
[49]Olsen D, Jiang J, Chang R, Duffy R, Sakaguchi M, Leigh S, Lundgard R, Ju J, Buschman F, Truong-Le V, Pham B, Polarek JW. Expression and characterization of a low molecular weight recombinant human gelatin:development of a substitute for animal-derived gelatin with superior features[J]. Protein. Expr. Purif.,2005,40:346-357
[50]Chalfie M. Green fluorescent protein[J]. Photochem. Photobiol.,1995,62: 651-656
[51]Cereghino GPL, Cereghino JL. Non-repressing carbon sources for alcohol oxidase AOX1 promoter of Pichia pastoris[J]. Curr. Opin. Biotech.,2002,13:329-332
[52]Kivirikko KI, Helaakoski T, Tasanen K, Vuori K, Myllyla R, Parkkonen T, Pihlajaniemi T. Molecular biology of prolyl 4-hydroxylase[J]. Ann. N. Y. Acad. Sci.,1990,580:132-14
[53]Kozak M. Structural features in eukaryotic mRNAs that modulate the initiation of translation[J]. J. Biol. Chem.,1991,266:19867-19870
[54]宋小岩,黄雅钦,黄明智.明胶应用技术某些新进展[J].明胶科学与技术,2002,22(1)：1-6
[55]Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes:gene splicing by overlap extension[J]. Gene.,1989,77:61-68
[56]Warrens AN, Jones MD, Lechler RI. Splicing by overlap extension by PCR using asymmetric amplification:an improved technique for the generation of hybrid proteins of immunological interest[J]. Gene.,1997,186:29-35
[57]萨姆布鲁克. 分子克隆实验指南[M].北京：科学出版社,1992
[58]陈红英,崔保安,李新生.融合基因mChIL-18/mChIFN-α克隆及其原核表达载体的构建[J].农业生物技术学报,2007,15(3)：388-392
[59]帅兴华,王少强,宋增峰,谢益民.明胶应用性能的研究[J].皮革化工,2007,24(3)：20-23
[60]Resina D, Cos O, Ferrer P, Valero F. Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 Promoter [J]. Biotechnol. Bioeng.,2005,91:760-767
[61]Pihlajaniemi T, Helaakoski T, Tasanen K, Myllyla R, Huhtala ML, Koivu J, Kivirikko K. Molecular cloning of the β-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene[J]. Embo. J.,1987,6:643-649
[62]Helaakoski T, Vuori K, Myllyla R, Kivirikko K, Pihlajaniemi T. Molecular cloning of the α-subunit of human prolyl 4-hydroxylase:The complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts[J].Proc. Natl.Acad. Sci.,1989,86:4392-4396
[63]Bruin EC, Werten MWT, Laane C. Endogenous prolyl 4-hydroxylation in Hansenula polymorpha and its use for the production of hydroxylated recombinant gelatin[J].Fems. Yeast. Res.,2002,1:291-298
[64]Vaughan PR, Galanis M, Richards KM, Tebb TA, Ramshaw JAM, Werkmeister JA. Production of Recombinant Hydroxylated Human Type Ⅲ Collagen Fragment in Saccharomyces cerevisiae[J]. DNA. Cell. Biol.,1998,17:511-518
[65]Kivirikko KI, Myllyharju J. Prolyl 4-hydroxylases and their protein disulf ide isomerase subunit[J]. Matrix.Biol.,1998,16:357-368
[66]Gelse K, Poschl E, Aigner T. Collagens-structure, function, and biosynthesis[J]. Adv. Drug. Deliver. Rev.,2003,55:1531-1546
[67]Bulleid NJ, John DCA, Kadler KE. Recombinant expression systems for the production of collagen[J]. Biochem. Soc. Trans.,2000,28:350
[68]Huang J, Foo CWP, Kaplan DL.Biosynthesis and application of silk-like and collagen-like proteins[J]. Polymer. Rev.,2007,47:29-62
[69]Myllyharju J, Nokelainen M, Vuorela A, Kivirikko KI. Expression of recombinant human type Ⅰ-Ⅲ collagens in the yeast Pichia pastoris[J]. Biochem. Soc. T.,2000,28:353-357
[70]Sreekrishna K, Brankamp RG, Kropp KE. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris[J]. Gene.,1997,190:55-62
[71]Wagner K, Poschl E, Turnay J, Balk JM. Coexpression of α and β subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen[J]. Biochem. J.,2000,352:907-911
[72]Gunnink IK, Vuorela A, Myllyharju J. Accumulation of properly folded human type Ⅲ procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris[J]. Matrix. Biol.,2000,19:29-36
[73]Pakkanen O, Pirskanen A, Myllyharju J. Selective expression of nonsecreted triple-helical and secreted single-chain recombinant collagen fragments in the yeast Pichia pastoris[J]. J. Biotechnol.,2006,123:248-256
[74]Murray WD, Duff SJB, Lanthier PH. Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris[J]. Appl. Microbiol. Biotechnol.,1989,32:95-100
[75]王镜岩,朱圣庚,徐长法.生物化学[M].北京：高等教育出版社,2002,215
[76]Lin YK, Liu DC. Comparison of physical-chemical properties of type Ⅰ collagen from different species[J]. Food. Chem.,2006,99:244-251