Multiresponsive Synthetic Cells, Precise Protein Micropatterns and Streptavidin-Biotin Conjugates Using the Interaction Between Ni2+-NTA and His-tagged Proteins
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Abstract
The binding of polyhistidine tags (His-tags) in proteins to Ni2+-NTA complexes is widely used in proteins purification, biofunctionalization of surfaces and protein modification. The small size of the tag, the mild conditions of the interaction (neutral pH and in the presence of salts) as well as reversibility in the presence of chelators and lowered pH allow preserving protein activity. In this thesis, these advantageous properties of the interaction between His-tags and Ni2+-NTA complexes were used to produce multiresponsive minimal synthetic cells, precise protein micropatterns and stoichiometrically well-defined streptavidin-biotin conjugates.
Minimal synthetic cells are cell-like compartments, which have been assembled from molecular building blocks and mimic certain functions of living cells. In chapter 2, a minimal synthetic cell which combines a multistimuli sensitive adhesion unit with an energy conversion module is developed, such that it can adhere to places that have the right environmental parameters for its ATP production. The multistimuli sensitive adhesion unit can sense environmental stimuli including light, pH, oxidative stress and the presence of metal ions and can regulate the adhesion of the synthetic cell to a substrate in response to these stimuli following a chemically coded logic. The adhesion unit is composed of the light and redox responsive protein interaction of iLID and Nano as well as the pH sensitive and metal ion mediated binding of protein His-tags to Ni2+-NTA complexes. The multistimuli responsive adhesion unit allows synthetic cells to self-position themselves in places under blue light illumination and no oxidative stress, with neutral pH and the presence of metal ions and carry out their light to ATP conversion function. Introducing such multiresponsive self-positioning module to synthetic cell is an important step towards their autonomy and transferable to other types synthetic cells.
The precise micropatterns provide structural and functional advantages in vast technological applications as well as fundamental research. In chapter 3, I developed a method of surface patterning proteins and cells with high spatiotemporal control using green light. A layer-by-layer (LbL) protein film with a green light cleavable protein, CarH, in the first layer is produced based on Ni2+-NTA-His-tag interaction. This enables the remote release of proteins in the upper layers by exposing the film to green light with 1 µm spatial and 10 s temporal resolution. The use of green light and the specific protein interactions overcome the current limitations of UV-light and unspecific protein immobilization, which can lead to protein denaturation. Green light lithography is successfully used to produce complex patterns of different functional His-tagged proteins including fluorescent proteins as well as the cell adhesion protein fibronectin.
In chapter 4, as a further step, two approaches for regulating cell adhesions in space and time with high precision have been developed based on the photocleavable CarH and the cell adhesion peptide, RGD. In the first design, which is call GREEN-ON, a protein layer of CarH was used to mask RGD, which is exposed upon green light illumination. In the second design, GREEN-OFF, the RGD sequence was integrated into the CarH protein and the protein CarH-RGD was used in the research of cell adhesion. Both designs allow for photoregulation and open new possibilities to investigate the dynamical regulation of cell adhesions.
Streptavidin-biotin conjugates with precise stoichiometries are powerful for the study of molecular biology, drug delivery and biotechnology. In chapter 5, I developed an approach to form monofunctional streptavidin conjugates with precise stoichiometries and number of open binding pockets. This method relies on an iminobiotin-polyhistidine tag, which allows separating streptavidin conjugates with different numbers of tags on a Ni2+-NTA column, and later reopening binding pockets at lowered pH to introduce a second functionality. Pure fluorescently labelled mono-, di- and trivalent streptavidin-biotin conjugates prepared in this way were used for imaging biotinylated cell surface molecules with controlled clustering. Moreover, these conjugates were functionalized with a second biotinylated molecule, folic acid-biotin, to investigate the importance of multivalent binding in targeted delivery of cells.
Building on this chemistry, I prepared stoichiometrically precise tetrafunctional streptavidin conjugates in chapter 6. An exemplary streptavidin conjugate with exactly one fluorescent label, one cell targeting group, one cell penetrating peptide and one drug demonstrates how each functionality contributed to overall efficacy of the drug. Such precise tetrafunctional streptavidin conjugates opens the door for combinatorial multifunctional libraries based on the well-established biotin-streptavidin interaction.
In summary, the interaction between Ni2+-NTA and His-tagged proteins is a reliable chemistry, which can be applied in many contexts as shown here in the design of stimuli-responsive synthetic cells, precise protein micropatterns and streptavidin-biotin conjugates. Our work provides a potential approach for the study of chemical biology, synthetic biology and cell biology.