Srivastava Atul K, Sharma Yogendra, Chary Kandala V R
Department of Chemical Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai 400005, India.
Protein Expr Purif. 2008 Apr;58(2):269-74. doi: 10.1016/j.pep.2007.12.010. Epub 2008 Jan 3.
A gene which encodes a hypothetical protein of 40 kDa has been identified in the genome of a marine bacterium Hahella chejuensis, as a putative member of betagamma-crystallin superfamily. This hypothetical protein contains a putative betagamma-crystallin-like domain, along with other domains for carbohydrate binding regions. It is named as Hahellin. A PCR amplified stretch of 92-amino acid residue long protein was cloned into pET21a vector and overexpressed in Escherichia coli strain BL21(DE3)pLysS cells. The recombinant Hahellin, produced as inclusion bodies, was estimated to be around 50% of the total cellular protein content which was solubilized in 8 M urea. The protein was purified and refolded using an anion exchange column. The MALDI-TOF mass spectrometry revealed the purity and monomeric nature of the protein. Further, a method to prepare isotopically (15N/13C) labeled protein with high yield for NMR studies is reported. The uniformly 15N-labeled Hahellin thus produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The well, dispersed peaks in the spectrum confirm that the protein is indeed well folded and suitable for further studies by NMR.
在海洋细菌济州海杆菌（Hahella chejuensis）的基因组中鉴定出一个编码40 kDa假定蛋白的基因，它被认为是βγ-晶状体蛋白超家族的一个成员。这个假定蛋白包含一个假定的βγ-晶状体蛋白样结构域，以及其他碳水化合物结合区域的结构域。它被命名为海杆菌蛋白（Hahellin）。一段经PCR扩增的92个氨基酸残基长的蛋白被克隆到pET21a载体中，并在大肠杆菌BL21(DE3)pLysS菌株中过量表达。以包涵体形式产生的重组海杆菌蛋白估计占总细胞蛋白含量的50%左右，该蛋白可在8 M尿素中溶解。使用阴离子交换柱对该蛋白进行纯化和复性。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）显示了该蛋白的纯度和单体性质。此外，还报道了一种用于核磁共振（NMR）研究的高产率制备同位素（15N/13C）标记蛋白的方法。通过记录灵敏度增强的二维[15N]-[1H]异核单量子相干（HSQC）谱对如此产生的均匀15N标记海杆菌蛋白进行了表征。谱图中峰的良好分散证实该蛋白确实折叠良好，适合通过核磁共振进行进一步研究。
