Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Biotechnol Bioeng. 1991 Aug 5;38(4):397-412. doi: 10.1002/bit.260380410.
Earlier experiments in our lab investigated the metabolic limitations of cloned-gene expression in bacterial cells (for over-production of beta-lactamase). These experiments showed that the steady-state concentration of ribosomal RNA decreased upon plasmid amplification while both the synthesis rate and steady-state beta-lactamase mRNA level increased significantly. This appeared to indicate substantial limitation exist within the transnational machinery of the bacterial cell at high copy numbers. To establish the generality of this phenomenon, the impact increasing protein expression from pa plasmid by chemically inducing a strong promoter while maintaining constant copy number has been investigated. A plasmid has been constructed which contains the lacZ gene under control of the tac promoter and contains the parB stability locus to maintain plasmid stability. Using this vector, beta-galactosidase expression in chemostat cultures operated at specific growth rates of 0.6 h(-1) was induced with IPTG such that enzyme activity was varied over a 460-fold range. When fully induced beta-galactosidase protein production represented 14 wt % of total cell protein. As transcription was induced, the synthesis rate of the beta-galactosidase mRNA increased 42-fold while the steady-state level of beta-galactosidase mRNA increased only fourfold. This indicates stability may play a larger role for beta-galactosidase expression with a strong promoter than seen with beta-lactamase production in the elevated copy number system. Furthermore, rRNA synthesis rates increased at high expression rates as seen in the copy number experiments. However, unlike the amplified-plasmid system, the steady-state levels of rRNA increased as well. Since the total protein levels closely followed the steady-state level of eRNA, transnational limitations are again suggested for the chemically induced transcription system.
早期我们实验室的实验研究了在细菌细胞中克隆基因表达的代谢限制（用于β-内酰胺酶的过度生产）。这些实验表明，当质粒扩增时，核糖体 RNA 的稳态浓度降低，而合成速率和稳态β-内酰胺酶 mRNA 水平显著增加。这似乎表明在高拷贝数下，细菌细胞的跨国机器存在实质性的限制。为了确定这种现象的普遍性，我们研究了通过化学诱导强启动子来增加质粒表达，同时保持恒定拷贝数对增加蛋白质表达的影响。构建了一个质粒，该质粒在 tac 启动子的控制下包含 lacZ 基因，并包含 parB 稳定性位点以维持质粒稳定性。使用该载体，在特定生长速率为 0.6 h(-1) 的恒化器培养物中，用 IPTG 诱导 lacZ 基因的表达，使酶活性在 460 倍的范围内变化。当完全诱导时，β-半乳糖苷酶蛋白的产量占总细胞蛋白的 14wt%。随着转录的诱导，β-半乳糖苷酶 mRNA 的合成速率增加了 42 倍，而β-半乳糖苷酶 mRNA 的稳态水平仅增加了 4 倍。这表明，与在高拷贝数系统中生产β-内酰胺酶相比，在强启动子诱导下，β-半乳糖苷酶表达的稳定性可能起着更大的作用。此外，正如在拷贝数实验中看到的那样，在高表达率下 rRNA 的合成速率增加。然而，与扩增质粒系统不同，rRNA 的稳态水平也增加了。由于总蛋白水平密切跟随 eRNA 的稳态水平，因此再次表明化学诱导转录系统存在跨国限制。
