生物工程學(xué)報(bào) Chin J Biotech 2008, January 25; 24(1): 21-26

摘 要: 為高效表達(dá)抗菌肽GK1并避免GK1的高抗菌活性對(duì)大腸桿菌宿主菌的致命影響, 將經(jīng)改造后的人胰島素原(mhPI)與GK1的融合基因(mhPI-GK1)克隆到表達(dá)載體pET28a中, 構(gòu)建出表達(dá)質(zhì)粒pET28a-mhPI-GK1, 轉(zhuǎn)化至大腸桿菌BL21(DE3)中進(jìn)行表達(dá)。融合蛋白在大腸桿菌中以包涵體形式表達(dá), 表達(dá)量占菌體總蛋白的20%。經(jīng)CNBr裂解,、陽(yáng)離子交換層析和RP-HPLC純化后, 每升發(fā)酵液可獲得5.7 mg純度大于97%的重組GK1,。質(zhì)譜檢測(cè)顯示重組GK1的分子量為2794.0 D, 抑菌活性實(shí)驗(yàn)表明純化后的重組GK1和化學(xué)合成GK1具有相同的抗菌活性。為利用基因工程方法大規(guī)模生產(chǎn)GK1奠定了基礎(chǔ),。

關(guān)鍵詞: 抗菌肽, 融合蛋白, 大腸桿菌

Fusion Expression of the Antimicrobial Peptide GK1 in Escherichia coli
Hairong Lu1,2, Guodong Li1, Hongyu Wu1, Jinjiang Huang1, Shilong Wang2, and Qingshan Huang1,3
1 Shanghai HI-Tech United Bio-Technological R&D Co. Ltd., Shanghai 201206, China 2 Institute of Protein Research, Tongji University, Shanghai 200092, China 3 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, China

Abstract: The antimicrobial peptide GK1, a derivative of Cecropin A, shows high antimicrobial activities against Gram-positive and Gram-negative bacteria. To avoid the lethal effects on Escherichia coli host cells during its expression, the GK1 gene was com-plexed with a modified human proinsulin (mhPI) gene, inserted into the vector pET28a to construct the recombinant expression plasmid (pET28a-mhPI-GK1) and then transformed into BL21(DE3). A fusion protein was expressed and resulted in insoluble inclu-sion bodies counting 20% (W/W) of total cellular proteins. Recombinant GK1 was cleaved from the mhPI-GK1 fusion protein by cyanogen bromide (CNBr) and purified by cation exchange chromatography and reverse-phase HPLC. The final concentration of recombinant GK1 was 5.7 mg/L Escherichia coli culture and the purity was above 97%. Its molecular weight measured by Electros-pray Ionizsation Mass Spectrometry (ESI-MS) was 2794 D, similar to that of the synthetic GK1. In addition, they have similar anti-bacterial activity. The results demonstrated that mhPI was capable of mediating high-level GK1 gene expression.
Keywords: antimicrobial peptide, fusion protein, Escherichia coli

全文鏈接：抗菌肽GK1在大腸桿菌中的融合表達(dá)

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