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首页> 外文期刊>Biotechnology Progress >Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry
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Expression System for Synthesis and Purification of Recombinant Human Growth Hormone in Pichia pastoris and Structural Analysis by MALDI-ToF Mass Spectrometry

机译：毕赤酵母中重组人生长激素的合成，纯化表达系统及MALDI-ToF质谱分析结构

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摘要

An expression system in Pichia pastoris for the production and purification of recombinant human growth hormone(rHGH)was designed and implemented.hGH cDNA sequence was cloned into pPICZaA vector under the control of AOX1 promoter,which included a polyhistidine-tag on the amino terminal end to enable affinity purification and a target site for Factor Xa protease such that protease cleavage in vitro would produce rhGH without any non-native N-and C-termini.Analyses of the affinity-purified rhGH product by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry(MALDI-TOF MS)showed a spectral peak at m/z 23699.Purified product digested with Factor Xa protease had a molecular mass of 22132 kDa.The molecular mass difference before and after Factor Xa protease digestion expectedly corresponds to the 12 amino acids in the rhGH amino terminus,which includes the EcoRI digestion site(Glu-Phe),the 6xHis tag for affinity purification,and the Factor Xa protease recognition sequence(Ile-Glu-Gly-Arg),a result that also indicates that the signal peptide was properly processed by P.pastoris.N-Terminal sequence analysis of the Factor Xa protease trimmed recombinant product confirmed the mature hGH sequence.Thus,the system designed functioned with its intended purpose effectively in expression,cleavage,and purification of the recombinant product.
机译：设计并实现了一种用于生产和纯化重组人生长激素（rHGH）的毕赤酵母表达系统。在AOX1启动子的控制下，将hGH cDNA序列克隆到pPICZaA载体中，该序列在氨基末端带有一个多组氨酸标签。以实现亲和纯化和因子Xa蛋白酶的靶位点，从而在体外裂解蛋白酶将产生rhGH，而没有任何非天然的N和C末端。通过基质辅助激光解吸/电离时间分析亲和纯化的rhGH产物飞行质谱（MALDI-TOF MS）在m / z 23699处显示一个光谱峰。用因子Xa蛋白酶消化的纯化产物的分子质量为22132 kDa，预期在因子Xa蛋白酶消化前后的分子量差相对应到rhGH氨基末端的12个氨基酸，包括EcoRI消化位点（Glu-Phe），用于亲和纯化的6xHis标签和因子Xa蛋白酶识别序列（Ile-Glu-Gly-Arg），也表明该信号肽已被巴斯德毕赤酵母正确加工。因子Xa蛋白酶修饰的重组产物的N末端序列分析证实了其成熟的hGH序列。设计的系统以其预期目的有效地在重组产物的表达，切割和纯化中起作用。

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