Can IPTG induce inclusion body proteins and increase concentration promote expression?

At 37 degrees Celsius, a trend can generally be observed within 3 hours, but inclusion body formation sometimes takes longer. I can do three points: 3h, 5h, and 6h. Note that IPTG itself has certain toxicity, especially when it is 1-2 mM, bacterial cell lysis and yield decrease over time. If the target band is weak after 3 hours, the temperature can be lowered to 30 degrees to induce 6 hours. The inclusion bodies may decrease but the bacterial volume may increase, resulting in a better overall yield. Your goal is to include bodies, and 37 degrees is more conducive to aggregation, but you need to closely monitor OD600. The decrease in OD indicates excessive induction and immediate collection of bacteria. 0.1 mM is too low, it is recommended to try at least 0.5 mM.

0.1 mM is indeed low, and it is not impossible to directly mention 1-2 mM. However, it is recommended to first perform gradients of 0.1, 0.3, 0.5, and 1.0 mM, fix ODs at 0.6-0.8 and 37 degrees, and take samples at 3h and 5h. Some proteins show an increase in total expression but a decrease in bacterial density at 1 mM, and the yield of inclusion bodies per unit volume may not necessarily be high. 2 mM is toxic to BL21 (DE3), and conventional inclusion body expression of 0.5-1 mM is generally sufficient unless the sequence is specific. You can first use a small shake flask to test the strength of the target band in the precipitate, and then enlarge it to 200 mL. The induction time can be checked for 3 hours, and if the target band is weak, it can be extended to 5-6 hours.

If expression remains low after increasing to 0.5-1 mM, concentration alone may be ineffective. 30 kD is not large in pET28a, but it is necessary to confirm the effects of rare codons in the target gene, mRNA secondary structure, or fusion tags. We have encountered proteins with very low expression in BL21(DE3), and after switching to Rosetta(DE3) or C43(DE3), the inclusion body yield improved markedly. Regarding induction time, 37°C for 3 hours is sufficient for most highly expressed inclusion body proteins, but 5-6 hours is more reliable for low-expression proteins. It is recommended to first perform a time gradient: 0.5 mM IPTG, 3 h, 5 h, and overnight, to assess the pellet amount. At the 200 mL scale, ensure sufficient antibiotic supplementation, and during induction, replacing with fresh medium can reduce toxicity.

Increasing IPTG concentration may increase total expression, but inclusion bodies can sometimes accumulate in large amounts within 3-4 h at 0.5 mM and 37 degrees. Since you have almost no supernatant at 0.1 mM and not much inclusion body either, this may not be due to IPTG concentration alone, but rather low expression of the protein itself. It is recommended to also check the plasmid sequence, promoter, RBS, and whether toxicity is causing plasmid loss. You can perform colony PCR on uninduced and induced cultures. During gradient induction, samples can be taken for SDS-PAGE to compare total protein, supernatant, and pellet. For a 200 mL shake flask, pay attention to the fill volume; too much liquid will lead to insufficient dissolved oxygen and can also affect expression. Use 200-250 rpm.