Ahuja Sanjay, Ahuja Satpal, Chen Qijun, Wahlgren Mats
Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, P, O, Box 280, Stockholm, S-17177, Sweden.
Malar J. 2006 Jun 25;5:52. doi: 10.1186/1475-2875-5-52.
Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally.
A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli.
Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1alpha (46.8%) > CIDR2beta (42.9%) > DBL2-4gamma (31.7%) > DBL2beta + C2 (30.6%) > DBL1alpha (24.9%) > DBL2-7epsilon (23.1%) > DBL2-5delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions.
The acidic terminal segment (ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.
恶性疟原虫红细胞膜蛋白抗原1（PfEMP1）引发的细胞相互作用是由多种DBL（达菲结合样）、CIDR（富含半胱氨酸的结构域间区域）和C2结构域类型介导的。这些结构域的功能和结构特征的阐明取决于可溶性重组蛋白的大量可得性。对3D7基因组株的PfEMP1结构域进行了先验预测，这些结构域最有可能以可溶性形式在大肠杆菌中表达，并通过实验得到了证实。
进行了一项计算分析，将序列依赖性特征与可溶性形式表达的可能性相关联，并通过菌落过滤印迹法验证预测结果，以快速鉴定大肠杆菌中可溶性蛋白的表达。
所有组成性PfEMP1结构域以可溶性形式表达的概率（平均溶解度百分比）从高到低的溶解度预测如下：ATS（56.7%）> CIDR1α（46.8%）> CIDR2β（42.9%）> DBL2 - 4γ（31.7%）> DBL2β + C2（30.6%）> DBL1α（24.9%）> DBL2 - 7ε（23.1%）> DBL2 - 5δ（14.8%）。结构域的长度与其在可溶性形式中成功表达的概率无关。检测可溶性蛋白的免疫印迹分析证实了溶解度预测的差异。
酸性末端片段（ATS）和CIDR α/β结构域类型适合在大肠杆菌中进行重组表达，而所有DBL亚型（α、β、γ、δ、ε）对于在大肠杆菌中重组表达获得可溶性蛋白而言是不佳选择。本研究对从事PfEMP1结构域功能和结构研究的人员具有参考价值。
