Revisiting rubisco as a protein substrate for insect midgut proteases

Area of Science:

• Biochemistry
• Insect Physiology
• Molecular Biology

Background:

• Understanding insect digestive proteases is crucial for pest management and insect biology.
• Rubisco large subunit (RBCL) is a key plant protein, but its use as a substrate for insect proteases is underexplored.
• Recombinant protein expression offers a standardized approach to obtaining consistent substrate material.

Purpose of the Study:

• To evaluate recombinant RBCL proteins as substrates for quantifying midgut protease activity in phytophagous Lepidoptera.
• To compare the efficacy of recombinant RBCL against casein and native Rubisco for protease assays.
• To assess the suitability of recombinant RBCL for in vitro enzyme assays and substrate zymography.

Main Methods:

• Cloning and expression of gene fragments encoding RBCL from host plants in Escherichia coli.
• Comparison of recombinant RBCL (rRBCL) with casein and native Rubisco as substrates for protease activity assays.
• Enzyme assays were conducted on fourth instar larvae of Helicoverpa armigera, Pieris brassicae, and Antheraea assamensis.

Main Results:

• Recombinant RBCL proteins, both cognate and non-cognate, performed similarly, highlighting conserved gene nature and substrate versatility.
• Casein and rRBCL generally outperformed native Rubisco as substrates, with exceptions noted when reducing agents were used.
• Similar total midgut protease activities were observed in Antheraea assamensis larvae feeding on different host plants, indicating efficient plant protein hydrolysis.

Conclusions:

• Recombinant RBCL is a suitable and versatile protein substrate for studying insect midgut proteases.
• The findings support the use of rRBCL in in vitro enzyme assays and substrate zymography for insect protease research.
• This approach provides a standardized method for investigating the digestive enzyme systems of herbivorous insects.