EXPRESSION AND CHARACTERIZATION OF WILD TYPE AND MUTANT PRES1 PEPTIDES AND HBSAG
野生型和突变型 PRES1 肽以及 HBSAG 的表达和表征
基本信息
- 批准号:3792646
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:Escherichia coli SDS polyacrylamide gel electrophoresis affinity chromatography binding proteins cell cycle proteins cell membrane chemical binding chimeric proteins gene expression hepatitis B antigens human tissue iodotyrosine liver cells molecular cloning monoclonal antibody mutant peptides plasmids protein sequence synthetic peptide tissue /cell culture
项目摘要
As described in the previous annual report, the more recent preparations of
our (wild type) recombinant preS1 peptide (rpreS1) contained two peptides
which are only separable by SDS-PAGE. When this rpreS1 preparation was
used as a labeled ligand by conjugating with 125 I-Bolton-Hunter reagent,no
specific binding to either plasma membranes prepared from human hepatocytes
or Hep G2 cells was observed. A sample of this rpreS1 preparation was sent
to Dr. Neurath but tested negative in its competition capability in his
unique binding system of HBsAg to HepG2 cells. Hence, point mutations were
performed and produced a mutant recombinant plasmid which can express a
fusion protein containing a 90 amino acid mutant preS1 peptide in E. coli.
Upon Factor Xa digestion, this mutant rpreS1 peptide, which contains tyr12
tyr13 rather than a wild type phe12 phe13 and a C-terminal gly90, was
released. The mutant rpreS1 has been purified by mono-Q column
chromatography, and 18 N-terminal amino acid residues have been confirmed
by amino acid sequence analysis. This mutant rpreS1 can be labeled with
carrier-free Na 125I by means of the Iodogen method because of the
availability of tyrosine residues. Binding studies are in progress.
如前一份年度报告所述,
我们的(野生型)重组preS 1肽(rpreS 1)含有两个肽
它们只能通过SDS-PAGE分离。 当这种rpreS 1制备被
通过与125 I-Bolton-Hunter试剂缀合用作标记配体,
与人肝细胞制备的质膜特异性结合
或Hep G2细胞。 将该rpreS 1制剂的样品
但在其竞争能力测试中呈阴性,
HBsAg与HepG 2细胞的独特结合系统。 因此,点突变是
进行并产生了突变重组质粒,其可以表达
在大肠杆菌中表达含有90个氨基酸的突变前S1肽的融合蛋白。杆菌
在Xa因子消化后,这种含有tyr 12的突变体rpreS 1肽
tyr 13而不是野生型phe 12 phe 13和C-末端gly 90,
发布 用mono-Q柱纯化了突变体rpreS 1
经高效液相色谱法纯化,得到18个N-末端氨基酸残基
通过氨基酸序列分析。 该突变体rpreS 1可以用
无载体Na 125 I通过Iodogen方法,因为
酪氨酸残基的可用性。 约束力研究正在进行中。
项目成果
期刊论文数量(0)
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会议论文数量(0)
专利数量(0)
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- 批准号:
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- 资助金额:
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