鼠羧肽酶原B在毕赤酵母中的表达、纯化与鉴定

Expression, Purification and Characterization of Rat Procarboxypeptidase B in Pichia pastoris

摘要:

为了在毕赤酵母中表达鼠羧肽酶原B（procarboxypeptidase B, proCPB）蛋白，以RT-PCR法从SD鼠胰腺细胞中克隆了proCPB基因，将其插入pPIC9载体，PEG1000介导转入毕赤酵母GS115细胞，在甲醇的诱导下，实现了proCPB在毕赤酵母中的成功表达。通过发酵条件的优化，使用BMGY(pH6.0)培养基，添加0.5％的酪蛋白水解物，于28℃，在起始OD₆₀₀达10.0时，每隔12h补加0.5％的甲醇，重组酵母GS115-proCPB表达的产物量可达到最高(500mg/L)，表达时间可达120h，表达的目的蛋白占总蛋白的94％以上。通过纯化条件的优化，采用两步疏水层析，可使目的蛋白的纯度达96％以上，蛋白得率达38％，重组proCPB活化后所得CPB的比活力可达110u/mg（CPB标准品为180u/mg）。相对分子量测定表明重组蛋白的分子量与理论值极相近，N-端氨基酸测序进一步表明proCPB基因在毕赤酵母中得到了正确的表达和翻译后加工修饰。

Abstract:

Carboxypeptidase B is a metalloenzyme, which is widely used for commercial and research purposes. Commercially available CPB purified from porcine or bovine pancreas is very expensive, and is not totally free from other proteases. In order to express the rat proCPB in Pichia pastoris, total RNA extracted from SD rat pancreas cells was reversely transcripted to synthesize cDNA, and the proCPB ORF was synthesized by PCR. After digestion with XhoⅠ and EcoRⅠ, the fragment was inserted into pPIC9, and the recombinant plasmid was named as p^PIC9-pro^CPB. By digestion with SacⅠ, the lined pPIC9-proCPB was transformed into Pichia pastoris strains GS115 with PEG1000 and integrated into their genomes. In the inducement of methanol, recombinant proCPB was successfully expressed in Pichia pastoris, and could be secreted into the supernatant in the culture. After optimizing the fermentation conditions, a higher production could be obtained when GS115-proCPB was induced in BMGY(pH6.0) at 28℃, with addition of 0.5% casein. The yield of recombinant protein reached 500mg/L, achieving over 94% of total protein in the culture supernatant. The purity of recombinant CPB can reach 96% after two step phenyl sepharose F F purification, and 38% of total protein can obtained after optimizing the pufication method. Comparing to the specific activity 180u/mg of CPB purchased from Sigma, the specific activity of recombinant CPB is 110u/mg. Mass spectrometry analyses showed the mass of the recombinant CPB was 35.1 kD, which is very close to the theory value 35.2 kD. Amino acid sequencing of N-terminal of recombinant CPB further indicated proCPB was expressed successfully and modificated correctly after translation.