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Cloning and sequencing of a human pancreatic tumor mucin cDNA 总被引:24,自引:0,他引:24
M S Lan S K Batra W N Qi R S Metzgar M A Hollingsworth 《The Journal of biological chemistry》1990,265(25):15294-15299
A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation. 相似文献
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Weiduan Xu Jianmin Chen Glenn Yamasaki John E. Murphy Baisong Mei 《Molecular biotechnology》2010,45(3):248-256
Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation
factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal
sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation
can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but
this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop
assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream
product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional
to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII
producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based
ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors
and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment.
To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing
bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation
between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be
rapidly performed, thereby making them effective for in-process monitoring of protein sialylation. 相似文献
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长裙竹荪Dictyophora indusiata是珍贵的食药用真菌,具有很强的抑菌作用,在天然防腐剂开发方面具有广阔的应用前景。本研究以长裙竹荪的抑菌活性为指标,通过萃取、3次不同流动相的硅胶柱层析、1次反相柱层析和薄层层析法对竹荪提取物进行分离纯化,得到一个抗菌活性强的单体化合物。根据核磁共振波谱等数据分析,推断该化合物为间苯三酚。以巨大芽孢杆菌和肠炎沙门氏菌为供试菌,用平板打孔法及原位抑菌法测定该化合物的抑菌效果,结果表明:该化合物对这两种菌有很强的抑制作用,半抑制浓度分别为83.06μg/mL和51.58μg/mL。本研究首次从长裙竹荪中获得具有抗菌活性的单体化合物间苯三酚,为竹荪天然抗菌物质的开发提供理论依据。 相似文献
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以糙皮侧耳Pleurotus ostreatus为材料,研究简单碳氮源及木质素纯品诱导条件对其木质纤维素酶活性的影响。结果表明,不同的碳源培养基和氮源培养基对糙皮侧耳漆酶活性、羧甲基纤维素酶活性和木聚糖酶活性均具有极显著的影响(P<0.001),且对糙皮侧耳菌丝生物量也有极显著的影响(P<0.001)。以蔗糖作主要碳源诱导物时,有利于提高糙皮侧耳漆酶活性;以果糖作主要碳源诱导物时,有利于提高糙皮侧耳羧甲基纤维素酶活性和菌丝生物量的积累;以葡萄糖作主要碳源诱导物时,有利于提高糙皮侧耳木聚糖酶活性。以酵母浸粉作主要氮源诱导物时,有利于提高糙皮侧耳漆酶活性和菌丝生物量的积累;以硝酸钾作为主要氮源诱导物时,有利于提高糙皮侧耳羧甲基纤维素酶活性;以硫酸铵作为主要氮源诱导物时,有利于提高糙皮侧耳木聚糖酶活性。碱性木素的存在,有利于提高糙皮侧耳漆酶活性,但不利于菌丝生物量的积累。与此同时,碱性木素的存在对糙皮侧耳羧甲基纤维素酶和木聚糖酶活性并没有促进作用。 相似文献
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