Determination of enzyme kinetic parameters of fast-acting Schizosaccharomyces pombe Ulp1 catalytic domain using Forster resonance energy transfer (FRET) assay.
The SUMO fusion technology has immensely contributed to the soluble production of therapeutics and other recombinant proteins in E. coli. The structure-based functionality of SUMO protease has remained the primary determinant for choosing SUMO as a solubility enhancer tag. This study details the quantification of kinetic parameters of commercially relevant S. pombe Ulp1 catalytic domain by employing a Forster resonance energy transfer (FRET) based assay. The energy transfer between the fluorophores allowed to elucidate the kinetic parameters precisely. For the FRET assay, the ECFP-SpSUMO-EYFP construct was successfully cloned in the pET28a vector. The fusion protein was efficaciously expressed and purified near homogeneity. The assay employed provided a real-time investigation of SpUlp1 catalysis. The enzyme turnover number (k cat ) was computed as 9.08 s -1 . The Michaelis-Menten constant, K M  was determined as 0.65 × 10 1  μM with a maximum velocity (V max ) of 0.045 μM/s. The substrate specificity ratio, k cat /K M  was calculated to be 1.39 × 10 6  M -1  s -1 . Using the FRET assay approach, the fast-acting nature of the SpUlp1 was analyzed in real-time at even 10 3  times higher molar substrate concentration. Thus, the kinetics of commercially relevant SpUlp1 was successfully demonstrated along with its large-scale production at 50 L bioreactor, where the maximum product concentration was 4.8 g/L. Additionally, the S. pombe SUMO used in the current study could potentially replace the S. cerevisiae SUMO as a solubility enhancer fusion tag.
Babbal SM, Khasa YP