重组人载脂蛋白C-I在毕赤酵母中的分泌表达及鉴定

目的:在毕赤酵母中高效分泌表达与天然人载脂蛋白C-I具有相同结构和活性的重组人载脂蛋白C-I( rhApoC-I).方法:RT-PCR法自人肝组织调取编码人ApoC-I的cDNA,构建真核分泌型表达载体pPICZα/hApoC-I.重组质粒线性化后转化毕赤酵母感受态细胞,甲醇诱导表达,建立rhApoC-I的毕赤酵母表达体系.对rhApoC-I进行Western blot分析和体外活性研究.结果:经PCR法克隆的hApoC-I cDNA序列与GenBank登录序列一致.SDS-PAGE和Western blot分析均在分子量约6.6kDa出现特异性条带,2L发酵条件下表达量达到80mg/L.结论:首次在毕赤酵母菌中高效分泌表达rhApoC-I,并确定其具有抑制血小板衍生生长因子诱导的平滑肌细胞增殖的抑制作用,为进一步研究其结构与功能提供物质基础.     

Arresten在毕赤酵母中的表达和鉴定

Arresten来自人Ⅳ型胶原α-1链非胶原末端,可抑制新血管生成。从人肝脏提取总RNA,RT-PCR扩增arres-ten的cDNA,T载体进一步扩增后与表达载体pPIC9连接,测序,确认后转入毕赤酵母,获得表达可溶性arresten的酵母细胞。表达产物经初步纯化后,用SDS-PAGE测定分子量为26kD,与理论计算值接近;表达产物对matrigel辅助的内皮细胞管化有明显抑制作用。上述结果表明用毕赤酵母表达了有活性的arresten。     

血小板衍生生长因子BB亚型在毕赤酵母中的表达及纯化

目的:用毕赤酵母表达系统表达重组人血小板衍生生长因子BB亚型(PDGF-BB)。方法:采用PT-PCR从人早幼粒白血病细胞系HL-60细胞中获得目的基因,克隆到表达载体p MEX9K中,质粒线性化后转化酵母表达菌株GS115,筛选后的酵母表达菌株经BMGY/BMMY培养基体系诱导表达后,通过疏水作用、离子交换、凝胶过滤纯化获得目的蛋白,采用SDS-PAGE、Western印迹、N端氨基酸序列和MTT增殖活性测定等方法检测目的蛋白性质及生物活性。结果:重组人PDGF-BB为分泌表达,表达量大于100 mg/L;经三步纯化,获得纯度高于95%且具有较高活性(5.0×105IU/mg)的目标蛋白。结论:利用毕赤酵母表达系统表达重组人PDGF-BB,表达产量高,成本低,工艺简单,易于工业化放大,有良好的市场前景。     

重叠延伸PCR法克隆人脂联素基因及其在毕赤酵母中表达

利用重叠延伸PCR法克隆出人脂联素基因, 连接至克隆载体pGEM-T中, 转化大肠杆菌DH5a, 通过测序对其进行序列分析后, 进一步构建毕赤酵母表达载体pPIC3.5K-ADPN, 通过电击法转化毕赤酵母GS115, 转化的重组酵母菌用甲醇诱导外源基因表达。经PCR、Southern blotting鉴定, 获得了重组毕赤酵母菌株; 经甲醇诱导人脂联素基因表达, Western blotting杂交鉴定证明人脂联素基因已经在毕赤酵母中成功表达。结果表明重叠延伸PCR法准确方便克隆出人脂联素基因, 在30oC, 1%甲醇诱导48 h, 人脂联素基因在巴斯德毕赤酵母中的表达最佳。     

III型类人胶原蛋白在大肠杆菌重组表达及发酵制备

【背景】胶原蛋白广泛应用于日用化工及生物医药中,相比传统方法,基因工程方法制备胶原蛋白具有避免病毒隐患、产量高等优点,逐步受到广泛关注。【目的】获得III型类人胶原蛋白基因,实现大肠杆菌中的异源表达。【方法】以人III型胶原蛋白α1链为模板,(Gly-X-Y)为最小研究单位,优选亲水性氨基酸,设计目标基因kit,构建重组大肠杆菌(Escherichia coli) pET-28a(+)-kit/BL21(DE3),并对其结构进行表征。【结果】类人胶原蛋白基因kit成功在大肠杆菌体系中表达,表达量约为0.53 g/L,7 L发酵罐上补料发酵后其最大表达量提高至3.02 g/L,亲和层析纯化类人胶原蛋白纯度约为91%,对其进行N端测序、氨基酸分析、质谱分析及圆二色谱分析,确定类人胶原蛋白成功表达。【结论】类人胶原蛋白的成功表达为未来规模化制备及其在日用化工及生物医药行业的应用奠定了基础。     

TIMP-2在毕赤酵母中的克隆与表达

为了克隆人基质金属蛋白酶组织抑制剂-2（TIMP-2）基因,并在Pichia pastoris中表达,根据GenBank上的TIMP-2的氨基酸序列和毕赤酵母偏爱密码子,通过化学合成和PCR相结合的方法获得了人的TIMP-2基因全长序列,构建了pPIC9-T2表达载体,电击转化到毕赤酵母,通过表型筛选和诱导表达得到蛋白表达工程菌,并对表达产物进行了分离纯化和生物学活性分析。     

利用毕赤酵母系统表达重组人生长激素的研究

为了获得重组人生长激素在毕赤酵母中高表达的菌株,按毕赤酵母基因密码子偏爱性,人工合成hGH的全基因序列.该基因被克隆到穿梭质粒pPIC9K中,PEG1000介导转入毕赤酵母GS115细胞,通过G418筛选获得高拷贝转化子.在甲醇的诱导下.实现了hGH在毕赤酵母中的成功表达.通过发酵条件的优化.发酵上清中的表达量达1537 mg/L经过超滤和两步层析,重组蛋白的得率这35%,纯度为97%,相对分子质量测定表明重组蛋白的相对分子质量与理论值相近.N-端氨基酸测序证实hGH基因在毕赤酵母中获得正确的表达.     

米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。     

抑制肿瘤生长活性的内皮生长抑制剂EDI-8t

对内源性的内皮生长抑制剂的序列来源分析后,用RT-PCR方法从人脐带中获得了417bp的cDNA,并克隆到毕赤酵母表达载体pPIC9,然后转化毕赤酵母GS115获得重组表达菌株。表达菌株通过甲醇诱导表达后,将发酵液通过分离纯化获得重组蛋白纯品,命名为EDI-8t。体外实验结果表明,这个胶原蛋白VIII来源的rhEDI-8t蛋白能特异性地抑制牛主动脉内皮细胞的增殖和迁移,并能引起内皮细胞的凋亡。动物体内实验结果表明,rhEDI-8t蛋白样品可有效抑制裸鼠皮下肝癌肿瘤的生长,抑制效果和参照品endostatin相同。但在小鼠黑色素瘤肺转移模型中,rhEDI-8t显示了高于参照品的抑癌效果。     

人内皮细胞抑制生长素在毕赤酵母中的高效表达

根据人胶原蛋白ⅩⅧNC结构域C末端编码内皮细胞抑制生长素 (Endostatin)的成熟多肽序列 ,人工合成了由毕赤酵母 (Pichiapastoris)中偏爱密码子组成的内皮细胞抑制生长素基因序列。该基因以N端融合的方式正确插入毕赤酵母诱导型表达载体pPICZαA中。通过电激将线性化的重组质粒转化到毕赤酵母SMD1 1 68细胞中 ,筛选获得表达具有生物活性的内皮细胞生长抑制素的高产菌株。在摇瓶发酵水平上 ,产量达到 80mg L。而利用生物反应器进行高密度发酵 ,Endostatin的产量可达 1 2 5mg L。纯化后的Endostatin具有抑制小鼠血管内皮细胞增殖的活性     

Cloning and sequencing of pro-alpha 1 (XI) collagen cDNA demonstrates that type XI belongs to the fibrillar class of collagens and reveals that the expression of the gene is not restricted to cartilagenous tissue

We have isolated several overlapping cDNA clones encoding alpha 1(XI) collagen chains from human and rat cDNA libraries. Together the human cDNAs code for 335 uninterrupted Gly-X-Y triplets, and a 264-amino acid C-propeptide, while the rat cDNAs cover the entire C-propeptide and about a third of the triple-helical domain. Comparison of the human and rodent nucleotide sequences showed a 95% sequence similarity. The identification of the clones as alpha 1(XI) cDNAs was based on the complete identity between the amino acid sequences of three human alpha 1(XI) cyanogen bromide peptides and the cDNA-derived sequence. Examination of and the cDNA-derived amino acid sequence showed a variety of structural features characteristic of fibrillar-forming collagens. In addition, nucleotide sequence analysis of a selected portion of the corresponding human gene revealed the characteristic 54-base pair exon motif. We conclude therefore that pro-alpha 1 (XI) collagen belongs to the group of fibrillar collagen genes. We also suggest that the expression of this gene is not restricted to cartilage, as previously thought, since the cDNA libraries from which the clones were isolated, originated from both cartilagenous and noncartilaginous tissues.     

The cloning and sequencing of alpha 1(VIII) collagen cDNAs demonstrate that type VIII collagen is a short chain collagen and contains triple-helical and carboxyl-terminal non-triple-helical domains similar to those of type X collagen

We have isolated two overlapping cDNA clones covering 2425 base pairs encoding a short type VIII collagen chain synthesized by rabbit corneal endothelial cells. The cDNAs encode an open reading frame of 744 amino acid residues containing a triple-helical domain of 454 residues flanked by 117- and 173-residue amino and carboxyl non-triple-helical domains (called NC2 and NC1, respectively). Based on the identity between the DNA-derived amino acid sequence and the amino acid sequence of a type VIII collagen CNBr peptide obtained from rabbit corneal Descemet's membrane, we conclude that the cDNAs code for a type VIII collagen chain. We give this chain the designation alpha 1(VIII). The alpha 1(VIII) triple-helical domain contains eight imperfections in the Gly-X-Y repeated structure with Gly-X instead of a full triplet. The length of the triple-helical domain and number and relative locations of these imperfections are remarkably similar to those of chicken alpha 1(X) collagen. The amino acid sequence of the carboxyl three-quarters of the NC1 domain has high sequence similarity to that of alpha 1(X) collagen. These data suggest that the triple-helix coding portions and carboxyl three-quarters of the NC1 domains of the alpha 1(VIII) and alpha 1(X) genes have a common evolutionary origin.     

Molecular cloning and partial characterization of a novel collagen chain, alpha 1(XVI), consisting of repetitive collagenous domains and cysteine-containing non-collagenous segments.

In an effort to identify new members of the collagen family, we screened a human placenta cDNA library with a collagenous probe. A novel 3.7 kb cDNA was identified encoding an open reading frame of 1,186 amino acids and containing a termination codon. The predicted polypeptide consists of 9 repetitive collagenous (stretches of Gly-X-Y) and several non-collagenous segments. Two cysteinyl residues separated by two amino acid residues (Cys-X-X-Cys) are regularly located in the N-terminal region of each non-collagenous segment. The deduced amino acid sequence described above is distinct from those of known types of collagen. Therefore, this novel collagen chain is designated alpha 1(XVI). Northern blot analysis revealed an alpha 1(XVI) mRNA of 5.2 kb, indicating that the overlapping cDNA clones isolated in this study covered nearly three-fourths of the mRNA. As a tool for further study on the expression of type XVI collagen, we prepared an antibody against the nonadecapeptide CFLSLERPRAEEARGDNSE, derived from the putative translation product of the cDNA. In immunoblot analysis, the antibody recognized a 160 kDa protein, which was bacterial collagenase-sensitive. Immunohistochemical stainings of human placental tissues with anti-peptide antibody revealed a positive reaction with amnion, the membranous tissue lining the amniotic cavity. The gene of alpha 1(XVI) chain, COL16A1, is mapped on the short arm of human chromosome 1 (1p13-p34).     

Sequence analysis of a full-length cDNA for the murine pro alpha 2(I) collagen chain: comparison of the derived primary structure with human pro alpha 2(I) collagen.

Comparison of the nucleotide sequence and primary structure of murine and human pro alpha 2(I) collagen indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-pro-peptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]338, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human pro alpha 2(I) collagen triple helices, with no truly nonconservative substitutions.     

The complete primary structure for the alpha 1-chain of mouse collagen IV. Differential evolution of collagen IV domains

We report here the complete nucleotide and amino acid sequences for the alpha 1-chain of mouse collagen IV which is 1669 amino acids in length, including a putative 27-residue signal peptide. In comparison with the amino acid sequence for the alpha 2-chain (Saus, J., Quinones, S., MacKrell, A. J., Blumberg, B., Muthkumaran, G., Pihlajaniemi, J., and Kurkinen, M. (1989) J. Biol. Chem. 264, 6318-6324), the two chains of collagen IV are 43% identical. Most of the interruptions of the Gly-X-Y repeat are homologously placed but strikingly show no sequence similarity between the two chains. Availability of the amino acid sequences for human collagen IV allows a detailed comparison of the primary structure of collagen IV and reveals evolutionarily conserved domains of the protein. Between the two species, the alpha 1 (IV) chains are 90.6% and the alpha 2 (IV) chains are 83.5% identical in sequence. We discuss these data with respect to differential evolution between and within the collagen IV chain types.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.     

Triple-helical peptides: an approach to collagen conformation, stability, and self-association

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.     

Complete primary structure of the alpha 1-chain of human basement membrane (type IV) collagen

We have determined the primary structure of the alpha 1(IV)-chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the alpha 1(IV)-chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy-terminal globular domain. The human alpha 1(IV)-chain contains a total of 21 interruptions in the collagenous Gly-X-Y repeat sequence. These interruptions vary in length between two and eleven residues. The alpha 1(IV)-chain contains four cysteine residues in the triple-helical domain, four cysteines in the 15-residue long noncollagenous sequence at the amino-terminus and 12 cysteines in the carboxy-terminal NC-domain.     

Sequence analysis of a full-length cDNA for the murine proα2(I) collagen chain: Comparison of the derived primary structure with human proα2(I) collagen

Comparison of the nucleotide sequence and primary structure of murine and human proα2(I) collage indicates a high degree of homology: 87% at the nucleotide level and 87% at the amino acid level, with the greatest degree of variability in the amino- and carboxy-propeptide domains. The homology is greatest in the triple helical domain, repeating [Gly-X-Y]₃₃₈, exhibiting 90% homology at the amino acid level, with only X and Y position residue substitutions. The X and Y residues show 86% homology between murine and human proα2(I) collagen triple helices, with no truly nonconservative substitutions.