Coupling transcription of a cloned gene to the lac operon with induction by isopropylthio-β-galactoside (IPTG) has been a favoured approach for recombinant protein expression using Escherichia coli as a heterologous host for more than six decades. Despite a wealth of experimental data gleaned over this period, a quantitative relationship between extracellular IPTG concentration and consequent levels of recombinant protein expression remains surprisingly elusive across a broad spectrum of experimental conditions. This is because gene expression under lac operon regulation is tightly correlated with intracellular IPTG concentration due to allosteric regulation of the lac repressor protein (lacY). An in-silico mathematical model established that uptake of IPTG across the cytoplasmic membrane of E. coli by simple diffusion was negligible. Conversely, lacY mediated active transport was a rapid process, taking only some seconds for internal and external IPTG concentrations to equalize. Optimizing kcat and KM parameters by targeted mutation of the galactoside binding site in lacY could be a future strategy to improve the performance of recombinant protein expression. For example, if kcat were reduced whilst KM was increased, active transport of IPTG across the cytoplasmic membrane would be reduced, thereby lessening the metabolic burden on the cell and expediating accumulation of recombinant protein. The computational model described herein is made freely available and is amenable to optimize recombinant protein expression in other heterologous hosts.

中文翻译：

---

IPTG（异丙硫基-β-半乳糖苷）跨大肠杆菌细胞质膜转运的机制——诱导重组蛋白表达的限速步骤

六十多年来，通过异丙硫基-β-半乳糖苷 (IPTG) 的诱导，将克隆基因的转录与 lac 操纵子偶联，一直是使用大肠杆菌作为异源宿主进行重组蛋白表达的首选方法。尽管在此期间收集了大量的实验数据，但在广泛的实验条件下，细胞外 IPTG 浓度与随后的重组蛋白表达水平之间的定量关系仍然令人惊讶地难以捉摸。这是因为由于 lac 阻遏蛋白 (lacY) 的变构调节，在 lac 操纵子调控下的基因表达与细胞内 IPTG 浓度密切相关。计算机数学模型表明，通过简单扩散穿过大肠杆菌细胞质膜的 IPTG 摄取可以忽略不计。相反，lacY 介导的主动转运是一个快速过程，只需几秒钟即可使内部和外部 IPTG 浓度达到平衡。通过 lacY 中半乳糖苷结合位点的靶向突变来优化 kcat 和 KM 参数可能是提高重组蛋白表达性能的未来策略。例如，如果kcat减少而KM增加，IPTG跨细胞质膜的主动转运就会减少，从而减轻细胞的代谢负担并加速重组蛋白的积累。本文描述的计算模型是免费提供的，并且适合于优化其他异源宿主中的重组蛋白表达。