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Insectherin – Production of statherins in insect cells

Our saliva contains natural proteins that can counteract tartar and biofilm formation. One promising protein is statherin. We want to use a biotechnological system to produce this protein cost-effectively and sustainably and analyse it with regard to its potential preventive properties.

Merlin Krause, Kristina Worch, Antje Burse (since July 2025)
REAHLIZE-funded project

Establishment of immunofluorescence detection of a GLUT6-like transport protein in insect cells

The establishment of a robust immunofluorescence staining protocol enables the analysis of the expression and distribution of membrane transporters in Sf9 cells. In addition, the methods developed in this project can be used to analyse other proteins produced by BEVS, which will benefit basic research and application-oriented biotechnological developments.

Upscaling baculovirus-mediated protein production

Upscaling cultivation and protein production from laboratory to reactor scale enables the expression of large quantities of target protein. One of its challenges is the formation of foam, which has a negative effect on cell growth, protein yield and process control. We were able to show that antifoam agents have a positive effect on the growth of Sf9 cells and can also increase protein production.

Optimisation of insect cell cultivation

We are constantly investigating strategies to improve the efficiency and reproducibility of Sf9 cell cultivation. One focus has been on modifying the surface properties of glass culture vessels. We were able to show that silanizing glassware has positive effects on cell growth, viability and experimental reproducibility on a small scale, as it minimises unwanted cell adhesion and aggregation.

Establishment of an mCherry reporter system

A reporter system using the fluorescent mCherry was established for microscopic control of baculovirus-mediated protein expression. This resulted in several constructs, both for the co-expression of mCherry with target proteins and for the expression of mCherry fusion proteins. The system has already been successfully applied, including for the fusion of mCherry to a membrane-bound protein, thus enabling the targeted investigation of localisation and expression in insect cell cultures.

Production and functional characterisation of an integral transport protein from the SLC2 family

Membrane transport proteins are crucial for the uptake and distribution of molecules in cells and organisms. Establishing a stable platform for the production of these difficult-to-express proteins enables the analysis of their biological properties and functionality, helping to identify potential biotechnological and medical applications. Within this project, a glucose (GLUT) transport protein from the solute carrier (SLC) family will therefore be produced.

Establishment of various methods for virus titer quantification

The multiplicity of infection (MOI) is of central importance for the course of infection and product yield. An MOI that is too low can lead to incomplete infection, while an MOI that is too high can result in premature cell death. Precise quantification of the virus concentration for calculating the MOI is therefore essential. In this context, mCherry- and GP64-based qPCR, a reliable plaque assay and a cell size-based assay were established.

Optimisation of transfection and infection of Sf9 cells

Efficient transfection and infection are crucial for the production of recombinant proteins using BEVS. Through the targeted optimisation of these processes – in both adherent and suspension cultures – virus yield, protein quality, protein yield, reproducibility and scalability can be significantly improved. A defined virus addition (MOI) and the correct harvest time can minimise cell stress, promote synchronous expression and increase protein expression. Precise infection control is essential for the successful use of the system in research and industry, especially in large-volume suspension systems.

Completed theses

Optimisation of transfection with PEI in insect cells. Bachelor's thesis (2024)

Optimisation of cultivation conditions and PEI transfection of Sf9 cells using recombinant bacmid DNA. Bachelor's thesis (2022)

Optimisation of cultivation and PEI transfection of Sf9 cells in suspension. Bachelor's thesis (2022)

Baculovirus-based production of an SLC2-like membrane transport protein in insect cells on a laboratory scale. Master's thesis (2022)

Optimisation of serum-free cultivation and transfection of Sf9 cells with baculoviral DNA using the mCherry reporter system. Bachelor's thesis (2019)

Optimisation of insect cell infection with baculoviruses using the mCherry reporter system. Bachelor's thesis (2019)

Establishment and upscaling of baculovirus-mediated protein expression in Sf9 cells. Master's thesis (2019)