摘要
Hepcidin是一个主要在肝脏表达的含8个半胱氨酸残基的小分子多肽,是机体铁稳态调控途径中关键性的效应分子,其主要生物学功能在于调节小肠上皮细胞对于铁的吸收。另外,hepcidin和许多富含半胱氨酸的抗菌肽类似具有显著的抗菌作用。本文采用大肠杆菌表达体系进行了人hepcidin的融合表达,并对重组hepcidin的生产工艺进行了研究。
根据hepcidin氨基酸序列和大肠杆菌密码子偏好性,化学合成了人hepcidin的基因序列,并在hepcidin编码序列的5′端引入了肠激酶特异性识别位点序列。我们首先构建了hepcidin的GST融合表达载体pGEX-hpc。尽管pGEX-hpc在25℃表达的GST-hepcidin 50%为可溶蛋白,但可溶蛋白不能采用谷胱甘肽Sepharose 4B亲和层析进行纯化。在载体pGEX-hpc的基础上构建了表达载体pET-hpc,pET-hpc表达的融合蛋白His-hepcidin大小为10.6kDa,N端带有6×His标签,并且其N端的担体序列中不含有半胱氨酸,hepcidin在His-hepcidin中所占比例为26.6%。采用His-hepcidin进行hepcidin的制备。
载体pET-hpc在E.coli BL(DE3)中表达的His-hepcidin融合蛋白以包涵体的形式存在。His-hepcidin诱导表达时,温度是影响蛋白表达水平和菌体生长的最主要因素。确定的His-hepcidin诱导表达条件为:含pET-hpc的E.coli BL(DE3)在37℃生长至OD600为0.6～0.8后加入0.2mmol/L IPTG于30℃诱导表达6h。表达后菌体收率为6.1g/L(湿重),His-hepcidin约占菌体总蛋白的25%。收获的菌体经超声破碎后离心分离His-hepcidin包涵体,采用1%浓度的Triton X-100洗涤2次,洗涤后包涵体收率为0.99g/L(湿重),其中包涵体中重组蛋白占总蛋白的76%,His-hepcidin产率为199mg/L。包涵体中半胱氨酸残基主要以还原形式存在,采用含100mmol/L β-ME的8mol/L尿素溶解包涵体。
Hepcidin is a low-molecular-weight, highly disulfide bonded peptide relevant to small intestine iron absorption and body iron homeostasis. Like other cysteine-rich antimicrobial peptides, hepcidin also exhibits obvious antibacterial and antifungal activity. In this paper we express 25 aa human hepcidin as fusion proteins in E. coli and develop a preparation route of recombinant hepcidin.According to codon preference in E. coli and amino acid sequence of hepcidin, the gene coding for hepcidin was synthesized which was combined a encoding sequence of enterokinase cleavage site at its 5' end. Firstly we constructed pGEX-hpc. Although about 50% of GST-hepcidin fusion protein expressed from pGEX-hpc was soluble after induction with 0.1 mM IPTG at 25 ℃, it could not be absorbed by Glutathione -Sepharose. On the basis of pGEX-hpc the plasmid pET-hpc was constructed. The proportion of hepcidin in His-hepcidin was about 26.6%, and the fusion protein contained an N-terminal 6> cells were lysed by sonication, the crude inclusion bodies were washed twice with 1% Triton X-100. The yield of washed inclusion body was 0.99g/L (wet weight), and the fusion protein in the inclusion body was 76%. Then the inclusion bodies was solubilized in 8mol/L urea containing lOOmmol/L 2-ME and 50mmol/L Tris-HCl(pH 8.0).Immobilized metal ion affinity chromatography (IMAC) was used to purify His-hepcidin in the condition of denaturation with two step elution. Firstly, 60mmol/L imidazole(pH 8.0) was used to elute the contaminants from the column. Then the elution of His-hepcidin was performed at pH 4.0. The binding capacity of Ni2+-IDA Sepharose Fast Flow for His-hepcidin was 30.4mg/ml resin.The disulfide bonds of hepcidin were formed before the fusion protein was cleaved by enterokinase. The oxidation of His-hepcidin was carried out in the cysteine-cystine system. The obtained His-hepcidin was diluted to a final protein concentration of 50(ig/mL with the buffer containing 3mol/L urea, 3mmol/L cysteine, 0.3mmol/L cystine and 20mmol/L Tris-HCl(pH8.0). Then the solution was kept at room temperature for 40h under moderate magnetic stirring. The monomer was up to 63% of total oxidized protein. The oxidized proteins were easily concentrated through IMAC and eluted with 300mmol/L imidazole containing 8mol/L urea. Then the dimeric and higher forms of the proteins were removed through Sephacryl S-100 HR gel filtration under denaturation condition at a flow rate of 0.46mL/min.We adopted continuous dilution to refold the monomer protein. Firstly the pooled monomer solution was adjusted to 100fi.g/mL His-hepcidin, 4mol/L urea, lOOmmol/L NaCl, 25mmol/L Tris-HCl, pH8.3. Then the solution was continuously diluted to 2mol/L urea and 50^xg/mL protein concentration within 8-1 Oh with refolding buffer(100mmol/L NaCl, 25mmol/L Tris-HCl, pH8.3). By this method, no protein precipitation was observed. The refolded protein was also concentrated with Ni-IDA-sepharose column and eluted with 300mmol/L imidazole
引文
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