Raftery M J, Collinson L, Geczy C L
Cytokine Research Unit, School of Pathology, University of New South Wales, Kensington, New South Wales, 2052, Australia.
Protein Expr Purif. 1999 Mar;15(2):228-35. doi: 10.1006/prep.1998.1015.
Recombinant murine MRP14 (mMRP14) was produced in Escherichia coli using the pGEX expression system. The mass of fusion protein, by electrospray ionization-mass spectrometry (ESI/MS), was 39,213 Da which compares well with the theoretical mass (39,210.4 Da). Thrombin digestion of fusion protein was expected at a cloned thrombin consensus sequence (. LVPRGS. ) located between glutathione S-transferase and mMRP14. Analysis of products of digestion by C4 reverse-phase HPLC and SDS-PAGE/Western blotting revealed two immunoreactive cleavage products with molecular weights around 13, 000. Masses of the two proteins determined by ESI/MS were 13,062 and 11,919 Da. The larger product corresponded to the expected mass of recombinant mMRP14 (13,061.9 Da). Analysis of the protein sequence of recombinant mMRP14 revealed a thrombin-like consensus sequence (. NNPRGH. ) located close to the C-terminus. The smaller protein corresponded to a truncated form of rec mMRP14 (rec MRP141-102) with a calculated mass of 11,918.6 Da. Optimization of the cleavage conditions resulted in >95% full-length rec mMRP14. Native mMRP14 contains one intramolecular disulfide bond between Cys79 and Cys90. The full-length recombinant protein was renatured and oxidized in ammonium acetate (pH approximately 7) for 96 h and formed >95% of the native intramolecular disulfide-bonded form. MRP141-102 bound substantially less 65Zn2+ compared to native mMRP14 or rec mMRP14 after transfer to polyvinylidene difluoride and incubation with 65ZnCl2, implicating the His residues located within the C-terminal domain in Zn2+ binding.
重组鼠源MRP14（mMRP14）是利用pGEX表达系统在大肠杆菌中产生的。通过电喷雾电离质谱（ESI/MS）测定，融合蛋白的质量为39213 Da，与理论质量（39210.4 Da）相当。预计融合蛋白在谷胱甘肽S-转移酶和mMRP14之间的克隆凝血酶共有序列（.LVPRGS.）处被凝血酶消化。通过C4反相HPLC和SDS-PAGE/蛋白质印迹法分析消化产物，发现了两种分子量约为13000的免疫反应性裂解产物。通过ESI/MS测定的这两种蛋白质的质量分别为13062和11919 Da。较大的产物对应于重组mMRP14的预期质量（13061.9 Da）。对重组mMRP14的蛋白质序列分析显示，靠近C端存在一个类似凝血酶的共有序列（.NNPRGH.）。较小的蛋白质对应于重组mMRP14（rec MRP141-102）的截短形式，计算质量为11918.6 Da。裂解条件的优化使得全长重组mMRP14的产率>95%。天然mMRP14在Cys79和Cys90之间含有一个分子内二硫键。全长重组蛋白在乙酸铵（pH约为7）中复性并氧化96小时，形成>95%的天然分子内二硫键结合形式。转移至聚偏二氟乙烯并与65ZnCl2孵育后，与天然mMRP14或重组mMRP14相比，MRP141-102结合的65Zn2+显著减少，这表明C端结构域中的组氨酸残基参与Zn2+结合。
