Heterologous expression, purification, and proteomic characterization of a 37 kDa pupal gut serine protease from Bombyx mori in Escherichia coli

• Published in: Journal of proteins and proteomics Vol. 15; no. 1; pp. 43 - 52
• Main Authors: Kannan, M., Kayalvizhi, N., Arunprasanna, V., Rameshkumar, N., Suganya, T., Krishnan, M.
• Format: Journal Article
• Language: English
• Published: Singapore Springer Nature Singapore 2024
• Subjects: Biological and Medical Physics; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biophysics; Cell Biology; Medicinal Chemistry; Proteomics; Systems Biology; Recombinant pupal gut serine protease; Mass spectrometry; Two-dimensional gel electrophoresis
• ISSN: 2524-4663; 2524-4663
• DOI: 10.1007/s42485-023-00126-4

Recently, there has been a growing interest in exploring the potential of insect proteases for industrial applications, owing to their versatile biochemical properties. The primary focus is on overexpressing a distinct 37 kDa pupal gut serine protease, termed PGSP, from
Bombyx mori
, using
Escherichia coli
(
E. coli
) as a heterologous system for the proteomic characterization and future industrial applications. The study involves amplifying the 987-base pair PGSP gene and incorporating it into the pET-30a ( +) expression vector, subsequently introduced into
E. coli
JM109. The fidelity of ligation is verified through restriction digestion, gene-specific PCR, and sequencing. The ensuing recombinant construct is transferred to
E. coli
BL21 (DE3), induced with IPTG to enable overexpression. Experimental results with varying IPTG concentrations (0.5–1.5 mM) confirm successful overexpression of recombinant PGSP within
E. coli
cells. SDS-PAGE reveals an overaccumulated protein band at approximately 42 kDa, present in both soluble and insoluble fractions. However, most of the PGSP is found to be insoluble, so we solubilize it using a denaturing buffer containing urea and then purify it using Ni–NTA agarose resin. Analysis of the purified recombinant PGSP via 2D-PAGE yields a distinct isoelectric point (p
I
) of 6.4. Notably, the observed molecular weight (
M
w
) discrepancy (42 kDa) from the expected 37 kDa is attributed to additional tags. In-gel solution and liquid chromatography and mass spectrometry confirm the protein as
Bombyx mori
p37k protease. The recombinant full-length PGSP, when overexpressed, exhibits a lack of protease activity due to its zymogenic state. This highlights the need for further research to identify the zymogen processing enzyme responsible for activating the protease, a subject that will be investigated in future studies.