Expression, Purification, and Characterization of Integrin Targeted Nanobody-SpyCatcher Fusion Proteins
Hossain, Md Hasibul (2025)
2025
Master's Programme in Biomedical Technology
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
Hyväksymispäivämäärä
2025-06-24
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202506247409
https://urn.fi/URN:NBN:fi:tuni-202506247409
Tiivistelmä
Integrins are cellular receptors that are involved in various cellular processes. Mediating cell adhesion is one of the most important functions of integrin. Cell adhesion of different cell types is important in the maintenance of tissue integrity and homeostasis. However, molecular mechanism of integrin activity is not fully understood. Hence, there’s a need for tools that can be used to study these important cellular receptors.
To target different cellular components, now-a-days nanobodies are utilized. Nanobodies are small antibody fragments derived from camelids. The small size of nanobodies, their tissue penetration and many other factors make them an attractive choice for specific recognition of cellular targets and an alternative to conventional antibodies. When nanobodies are genetically fused with SpyCatcher, it allows modular assembly of molecular tools via SpyTag/SpyCatcher isopeptide conjugation. SpyTag/SpyCatcher is a bioconjugation system which allows covalent conjugation between two molecules spontaneously in almost every condition, including otherwise harsh and unfavorable for other conjugation systems.
In this thesis, I designed nanobody-based tools consisting of nanobodies recognizing different integrin subunits i.e., αM, β1, and β2 genetically fused with the SpyCatcher protein. Targets were chosen based on the availability of nanobody sequences as well as their biological importance. The designed constructs were expressed in Escherichia coli and purified using Ni-NTA affinity chromatography combined with ion exchange and size exclusion chromatography. Biochemical properties of purified tools were studied utilizing standard techniques including SDS-PAGE, western blot, dynamic light scattering, and mass spectrometry. The functionality of the bioconjugation and specificity of integrin recognition was also studied. Bioconjugation was tested utilizing band-shift assay. Target integrin recognition was evaluated in cell-based experiments using THP-1 derived macrophage cells. This specific cell line was utilized because it expresses all the target integrins.
Protein expression and purification were both successful. All the Nanobody-SpyCatcher (NbSC) proteins were soluble and biochemical tests indicated those were well behaving. SpyCatcher moieties in the purified tools were confirmed to be reactive. In cell-based experiments, at least one of the NbSCs was confirmed to be functional in the recognition of its specific target. The other NbSCs will still need further evaluation to confirm their functionality. These newly developed NbSCs have the potential to uncover new biological insights in integrin research.
To target different cellular components, now-a-days nanobodies are utilized. Nanobodies are small antibody fragments derived from camelids. The small size of nanobodies, their tissue penetration and many other factors make them an attractive choice for specific recognition of cellular targets and an alternative to conventional antibodies. When nanobodies are genetically fused with SpyCatcher, it allows modular assembly of molecular tools via SpyTag/SpyCatcher isopeptide conjugation. SpyTag/SpyCatcher is a bioconjugation system which allows covalent conjugation between two molecules spontaneously in almost every condition, including otherwise harsh and unfavorable for other conjugation systems.
In this thesis, I designed nanobody-based tools consisting of nanobodies recognizing different integrin subunits i.e., αM, β1, and β2 genetically fused with the SpyCatcher protein. Targets were chosen based on the availability of nanobody sequences as well as their biological importance. The designed constructs were expressed in Escherichia coli and purified using Ni-NTA affinity chromatography combined with ion exchange and size exclusion chromatography. Biochemical properties of purified tools were studied utilizing standard techniques including SDS-PAGE, western blot, dynamic light scattering, and mass spectrometry. The functionality of the bioconjugation and specificity of integrin recognition was also studied. Bioconjugation was tested utilizing band-shift assay. Target integrin recognition was evaluated in cell-based experiments using THP-1 derived macrophage cells. This specific cell line was utilized because it expresses all the target integrins.
Protein expression and purification were both successful. All the Nanobody-SpyCatcher (NbSC) proteins were soluble and biochemical tests indicated those were well behaving. SpyCatcher moieties in the purified tools were confirmed to be reactive. In cell-based experiments, at least one of the NbSCs was confirmed to be functional in the recognition of its specific target. The other NbSCs will still need further evaluation to confirm their functionality. These newly developed NbSCs have the potential to uncover new biological insights in integrin research.