Krishna Nagampalli Raghavendra Sashi, Krushna N S A, Narayanan R B, Rajan S S, Gunasekaran K
CAS in Crystallography and Biophysics, University of Madras, Chennai 600 025, India.
Protein Expr Purif. 2011 Oct;79(2):245-50. doi: 10.1016/j.pep.2011.06.014. Epub 2011 Jul 1.
Bm-33 (pepsin inhibitor homolog) produced by the human filarial parasite Brugia malayi, was expressed in Escherichia coli. Expression of rBm33 in BL21 (DE3), Rosetta-2 gami (DE3) pLysS and GJ1158 bacterial strains, results in the accumulation of a 33 kDa protein in inclusion bodies. Inactive rBm-33 was purified under the denaturing conditions and refolded by step wise dialysis using buffers of pH ranging from 11 to 7. Size exclusion chromatography of rBm-33 (refolded) reveals that nearly 83% of the recombinant protein exhibits pepsin inhibition activity. Circular dichroism studies indicate that the protein is predominantly composed of 85% α-helix. rBm-33 (refolded) was assessed for its pepsin inhibition activity using casein agar plate method, UV-spectroscopy and zymogram analysis. These findings suggest that rBm-33 (refolded) has affinity for human pepsin and completely inhibits the proteolytic activity with the gradual increase in rBm-33 (refolded) concentration. Size exclusion chromatography reveals the formation of rBm-33-pepsin complex and was cross checked using immunoblot with glutaraldehyde cross linking. These findings reveal that rBm-33 (refolded) is in native fold to exhibit pepsin inhibition.
由人类丝虫寄生虫马来布鲁线虫产生的Bm - 33（胃蛋白酶抑制剂同源物）在大肠杆菌中表达。rBm33在BL21 (DE3)、Rosetta - 2 gami (DE3) pLysS和GJ1158菌株中表达，导致一种33 kDa的蛋白质在包涵体中积累。无活性的rBm - 33在变性条件下纯化，并使用pH范围从11到7的缓冲液通过逐步透析进行复性。rBm - 33（复性后）的尺寸排阻色谱显示，近83%的重组蛋白具有胃蛋白酶抑制活性。圆二色性研究表明，该蛋白质主要由85%的α - 螺旋组成。使用酪蛋白琼脂平板法、紫外光谱法和酶谱分析评估rBm - 33（复性后）的胃蛋白酶抑制活性。这些发现表明，rBm - 33（复性后）对人胃蛋白酶具有亲和力，并随着rBm - 33（复性后）浓度的逐渐增加而完全抑制其蛋白水解活性。尺寸排阻色谱显示形成了rBm - 33 - 胃蛋白酶复合物，并使用戊二醛交联的免疫印迹进行了交叉检查。这些发现表明，rBm - 33（复性后）处于天然折叠状态以表现出胃蛋白酶抑制作用。
