LACTOSE FED-BATCH OVEREXPRESSION OF RECOMBINANT METALLOPROTEINS IN ESCHERICHIA-COLI BL21(DE3) - PROCESS-CONTROL YIELDING HIGH-LEVELS OF METAL-INCORPORATED, SOLUBLE-PROTEIN
LACTOSE FED-BATCH OVEREXPRESSION OF RECOMBINANT METALLOPROTEINS IN ESCHERICHIA-COLI BL21(DE3) - PROCESS-CONTROL YIELDING HIGH-LEVELS OF METAL-INCORPORATED, SOLUBLE-PROTEIN
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DOI：
10.1006/prep.1995.1085
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发表时间：
1995-10-01
影响因子：
1.6
通讯作者：
KENEALY, WR
中科院分区：
文献类型：
--
作者：
HOFFMAN, BJ;BROADWATER, JA;KENEALY, WR
本发明描述了一种在中试规模发酵设备中使用葡萄糖补料分批初始生长，随后是葡萄糖和乳糖混合物的对数中期补料来生产重组蛋白的方法。使用宿主菌株大肠杆菌BL21（DE3），二铁蛋白硬脂酰-酰基载体蛋白Delta（9）去饱和酶已经以高达12g ×升（-1）干细胞重量的生物量水平过表达，这表示相对于从分批发酵获得的体积生产率，体积生产率增加了12倍。在这些条件下，最多36%的总细胞蛋白质积累为去饱和酶多肽。的补料分批培养，一个持续的，虽然较慢，诱导条件下的指数增长，和形成的可溶性全蛋白和包涵体之间的有利的分区之间的增长速度减慢的相关性报道。这种相关性表明，补料分批技术可以用于有益地影响过表达蛋白质成熟中的限速过程，如本文提出的金属摄取和掺入。通过使用由补料分批方法产生的细胞，含铁的可溶性去饱和酶可以以高达66 mg × g（-1）干细胞重量的产率（类似于500 mg ×升（-1）培养物）纯化，这表示相对于从分批发酵获得的产率，产率增加了3至5倍。此外，这些方法适用于在E. coli BL21（DE3）pLysS，一种用于过表达毒性蛋白的宿主菌株。(C)出版社：Academic Press
A method for producing recombinant proteins in pilot scale fermentation equipment using a glucose fed-batch initial growth, followed by a midlog phase feeding of a glucose and lactose mixture is described. Using the host strain Escherichia coli BL21(DE3), the diiron protein stearoyl-acyl carrier protein Delta(9) desaturase has been overexpressed at a biomass level of up to 12 g x liter(-1) dry cell weight, representing a 12-fold increase in volumetric productivity relative to that obtained from batch fermentations. Under these conditions, a maximum of 36% of the total cellular protein accumulates as the desaturase polypeptide. A correlation between the slowed growth rate of the fed-batch culture, a continued, albeit slower, exponential growth under inducing conditions, and a favorable partitioning between formation of the soluble holoprotein and inclusion bodies is reported. This correlation suggests that fed-batch techniques can be used to beneficially influence rate-limiting processes in the maturation of overexpressed proteins, such as metal uptake and incorporation proposed here. By using cells produced from the fed-batch method, the iron-containing, soluble desaturase can be purified in a yield of up to 66 mg x g(-1) dry cell weight (similar to 500 mg X liter(-1) culture), representing a three to fivefold increase in the yield relative to that obtained from batch fermentations. In addition, these methods are suitable for the production of the Anabena 7120 vegetative [2Fe 2S] ferredoxin in E. coli BL21(DE3) pLysS, a host strain used for the overexpression of toxic proteins. (C) 1995 Academic Press, Inc.