Spangfort M D, Ipsen H, Sparholt S H, Aasmul-Olsen S, Larsen M R, Mørtz E, Roepstorff P, Larsen J N
Research Department, ALK-ABELLO Group, Hørsholm, Denmark.
Protein Expr Purif. 1996 Nov;8(3):365-73. doi: 10.1006/prep.1996.0112.
A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site. A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage. Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa. Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence. Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping. The yield of purified recombinant Bet v 1 was 10 mg per liter E. coli cell culture. Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs. The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients. Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1. Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass. Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.
已克隆了来自疣皮桦（白桦）的编码花粉主要过敏原Bet v 1的基因，并使其在大肠杆菌中作为与麦芽糖结合蛋白及一个因子Xa蛋白酶裂解位点的融合体进行表达。设计了一种基于聚合酶链反应的通用克隆策略来定位因子Xa蛋白酶裂解位点，以便在裂解后产生Bet v 1的真实氨基末端。融合蛋白通过直链淀粉亲和层析进行分离，并通过与因子Xa孵育进行酶切。重组Bet v 1通过凝胶过滤进行分离，在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时呈现出一条表观分子量为17 kDa的单条带。对前20个氨基酸进行的N端测序显示与推导的Bet v 1 DNA序列完全一致。质谱分析表明重组Bet v 1的分子量为17,440±2 Da；用Lys-C消化并通过质谱肽图分析可验证86%的重组Bet v 1氨基酸序列。纯化的重组Bet v 1的产量为每升大肠杆菌细胞培养物10 mg。纯化的重组蛋白的二维凝胶电泳产生了一个主要蛋白点以及一两个聚焦在稍有不同pH值处的次要蛋白点。当使用一组鼠单克隆抗体和来自桦树花粉过敏患者的血清IgE进行比较时，重组蛋白的免疫化学特性与天然存在的Bet v 1无法区分。此外，重组Bet v 1引发的T细胞增殖与天然Bet v 1相当。因此，用于细菌表达和蛋白质纯化的方法可产生具有正确N端序列和分子量的折叠重组Bet v 1的相对高产率。此外，重组Bet v 1的B细胞和T细胞表位结构与来自花粉的天然蛋白非常相似。
