Motwani N, Talarico T, Jain S, Bajwa W, Blackburn R, Nwosu V, Holland M, DeAngelo J, Privalle C, Keng T
Apex Bioscience, Inc., Research Triangle Park, North Carolina 27709-2847, USA.
Protein Expr Purif. 1996 Dec;8(4):447-55. doi: 10.1006/prep.1996.0123.
Hemoglobin Rainier is a naturally occurring hemoglobin variant in which the beta 145 tyrosine is substituted with cysteine. The alpha and beta Rainier globin cDNAs were cloned in a high copy number vector and expressed in Saccharomyces cerevisiae under the control of galactose-regulated hybrid promoters. Using this system, we have expressed individual alpha and beta Rainier globin chains. Coexpression of both alpha and beta Rainier cDNAs resulted in the production of a functional hemoglobin molecule. Purification of the recombinant protein was accomplished by ion exchange chromatography. The N-termini of the alpha and beta chains were correctly processed, and the molecular mass, as determined by mass spectrometry, indicated amino acid composition identical to that of natural hemoglobin Rainier. The chromatographic properties of the recombinant hemoglobin Rainier were similar to human-derived hemoglobin A0. The purified recombinant hemoglobin molecule was shown to have an elevated oxygen affinity and a reduced cooperativity as previously reported for natural hemoglobin Rainier. Production of recombinant hemoglobin and especially hemoglobin variants like hemoglobin Rainier has the potential to facilitate use of hemoglobin as a blood substitute as well as in specific applications, such as for use as a therapeutic agent in the treatment of hypotension associated with septic shock.
雷尼尔血红蛋白是一种天然存在的血红蛋白变体，其中β145位的酪氨酸被半胱氨酸取代。α和β雷尼尔珠蛋白cDNA被克隆到高拷贝数载体中，并在半乳糖调节的杂交启动子控制下在酿酒酵母中表达。利用该系统，我们表达了单独的α和β雷尼尔珠蛋白链。α和β雷尼尔cDNA的共表达导致产生了一种功能性血红蛋白分子。重组蛋白通过离子交换色谱法进行纯化。α链和β链的N端被正确加工，质谱测定的分子量表明其氨基酸组成与天然雷尼尔血红蛋白相同。重组雷尼尔血红蛋白的色谱性质与源自人类的血红蛋白A0相似。纯化的重组血红蛋白分子显示出具有升高的氧亲和力和降低的协同性，这与先前报道的天然雷尼尔血红蛋白情况相同。重组血红蛋白尤其是像雷尼尔血红蛋白这样的血红蛋白变体的生产，有可能促进血红蛋白作为血液替代品的使用，以及在特定应用中的使用，例如用作治疗与感染性休克相关的低血压的治疗剂。
