Gill R T, Cha H J, Jain A, Rao G, Bentley W E
Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742, USA.
Biotechnol Bioeng. 1998 Jul 20;59(2):248-59.
The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.
在通气的重组大肠杆菌发酵中，通过调节添加还原剂二硫苏糖醇（DTT）实现了对培养物氧化还原电位（CRP）的独立控制。添加适量水平的DTT对特定氧摄取、生长速率和溶解氧的影响最小。添加过量的DTT对细胞有毒，导致生长停止。在分批发酵实验中，随着DTT添加量的增加，氯霉素乙酰转移酶（CAT）活性（纳摩尔/微克总蛋白分钟）降低。为了进一步研究影响CAT活性的机制，进行了实验以测定热休克蛋白表达和特定CAT活性（纳摩尔/微克CAT分钟）。发现添加DTT后，诸如GroEL和DnaK等分子伴侣的表达增加。此外，在添加DTT期间，σ因子32（σ32）和几种蛋白酶的表达显著增加。随着DTT的添加，特定CAT活性（纳摩尔/微克CAT分钟）变化很大，然而，在大肠杆菌RR1 [pBR329]和JM105 [pROEX-CAT]菌株中，在最高DTT添加水平下发现活性最低。同时，发现细胞应激在相同DTT水平时达到最大值。尽管添加DTT有可能直接影响细胞内蛋白质折叠，但细胞内应激增加所产生的影响可能是主要效应因素。在细胞胞质内测量到DTT的影响，这表明周质氧化还原电位也发生了改变。在生长速率和氧摄取变化最小的情况下，特定CAT活性、分子伴侣和其他热休克蛋白的变化表明，氧化还原电位的体外控制为影响通气大肠杆菌发酵中异源蛋白质的产量和构象提供了一种新方法。
