See Linkers for Affinity Chromatography
Types of Linkers:
Flexible Linkers:
Flexible linkers are usually applied when the joined domains require a certain deree of movement of interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (eg., Ser or Thr) amino aicds. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains.. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous wolutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaciton between the linker and the protein moieties. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369. )
Polypeptides that form suitable flexible linkers are well known int he art. Flexible linkers typically include glycicne, because this amino acid, which lacks a side chain, is unique in its rotational freedom. Serine or threonine can be interspersed in the linker to increase hydrophilicity. (Kim, US 2007/0042378).
Unstructured random coil peptide linkers may, but do not necessarily, comprise Gly-rich regions, ntoably unforlded character of any lenght or a combination thereof. (Lin, US 2016/0185826)
Rigid linkers:
While felxible linkers have the advantage to connect the functional domains passively and permitting certain degree of movements, the lack of regidity of these linkers can be a limitation. There are several examples wehre the use of flexible linkers resulted in poor expression yields or loss of biological activity. For instance, a Tf-granulocyte colony stimulating factor (G-CSF) fusion protein failed to be expressed with a felixble (GGGGS)c linker. In anotehr report, the immunoglobulin binding ability of protein G domain in a protein G-Vargula luciferase fusion protein was not recovered after interserting a flexible GGGGS linker. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369.
–Alpha helix-forming linkers: with the sequence of (EAAAK)n have been applied to the construction of many recombinant fusion proteins. Many natural linkers exhibit alpha helical structures. The structure is rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff alpha helical linkers may act as rigid spacers between protein domains. (Chen, “Fusion Protein Linkers: Property, Design and Funcitonality” Adv Drug Deliv Rev: 2013, October 15, 65(1); 1357-1369.)
—-Coil-Tag Platform: As the ADC field matures and demand for greater homogeneity and site-specificity intensifies, new bioconjugation platforms are emerging to address the limitations of classical cystine-maelimide and lysine-amid chemistries. The Coil-Tag platform is a modular, supramolecualr conjugaiton strategy based on the self-assembly of engineerred alpha-helical coiled-coil peptides. CoilTag mimics naturally found super-secondary structures in proteins consisting of two or more alpha-helical peptides. These structural motifs are ubiquitous mediators of protein-protein interactions, spanning eukaryotic transcription factor leucine zippers to structural proteins, such as keratin and myosin. Coiled coil conjugation relies on orthogonal complementary interactions between repeating peptide sequencings, where each amino acid residue plays a distinct role in governing the staiblity and affinity of the coil pair. Lending to its versatility, the particular peptide sequence can be either inspired by nature or designed de novo. For example, peptide sequences based on a heptad repeatfollow the pattern (abcdefg)n, where n represents the nubmer of repeats. Each alpha-helical turn spans 3.5 amino acid residues per turn, positioning a and d to face in toward each other, creating a hydrophobic core driven by Van der Waals interactions and pi-pi stacking. Through rational sequence design, one controls coiled coils’ parameters such as orientation (parallel vs. anti-parallel assembly), oligomerization state (dimeric, trimeric, or tetrameric assembly) and stimuli responsiveness to pH and temperature. Increasing the number of heptad repeats, n, enables precice control over binding stability, which incorproating covalent lactam staples within the peptidic backbone enhances mechanical stability. Ultimately, coiled coils are highly modular building blocks capable of supporting diverse, programmable assemblies across a wide range of bioconjugation applications. In support of coiled-coil conjugaiton for ADC and future coinical utility, Coil-tag is finding sues across syntehtic biology, diagnostics and materials science disciplines. For example, the split, universal, and programmable (SUPRA) CAR system for T cell therapy includes a coil on its surface that assembles with a complementary coil containing a targeting scFv, allowing for tumor-killing. (Grinstaff, “Coil-Tag conjguation: a next gnsite-specific ADC fabrication technology” Bioconjguate insights, 2026).
Rigid linkers may, but do not necessarily comprise at least one alpha-helical structure, Pro-rich sequence or a combination of both. (Lin, US 2016/0185826)
Clevage linkers:
Clevable linkers may, but do not necessarily, comprise at least one disulfide bond, thrombin-senstive sequence, protease-sensitive sequence or a combination thereof. (Lin, US 2016/0185826)
Non-Covalent Protein Adsorption
Non-covalent methods of protein immobilization are widely employed and involve either passive adsorption onto hydrophobic surfaces or electrostatic interactions with charged surfaces. Here, the use of nitrocellulose membranes or polystyrene microtiter plates for hydrophobic adsorption and polysine coated slides for electrostatic binding are known. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)
Covalent Immobilziation Methods
For more stable attachment, the formation fo covalent bonds is required which are generally formed through reaction with functional groups present on the protein surface. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)
Amide bonds: The exposed amine groups of Lys residues readily react with supports bearing active esters, with the most common being N-hydroxysuccinimide (NHS) esters to form stable amide bonds. A disadvantage of using NHS esters is that they are unstable in aqueous conditions and thus attachment of protoein in aqueous buffers will compete with ester hydrolysis, resulting in only modest immobilizaiton yeilds. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)
Thioether bonds: The Cys residue bearing the thiol group is often employed for protein immobilziation and readily ungergoes conjugage addtion tihe alpha2beta-unsaturated carbonyls (e.g., maleimides) to form stable thioether bonds. (Wong “Selective covalent protein immbilization: strategies and applications” Chem Rev 2009, 109 4025-4053)
Biotinylate proteins & immobilziation onto avidin-functioned substrate:
Streptavidin-biotin binding is a rapid, specific and can occur under conditions where most other ptoeins have denatured, wuch as high temperatures. A breakthrough for the sue of bitoin for protein modifciation was harnessing the cell’s natural machinery for bitoin conjugation, using the E. coli enzyme BirA to acheive precise biotin modificaiton. The natural substarate of BiA is the biotin carboxyl carrier protein (BCCP) requring fusion of at least 75 residues to the target prtoein. However, phage display selection enalbed the development of the AviTag which is superior to BCCp as a BirA substrate but only 15 mino acids in lenght, so estending the range of prtoein sites amenable to site-specific enzymatic biotinylation. (Methods Mol. Bio. 2015, 1266, 171-184).
Yao (Nature protocols, 1(5), 2006) discloses site-specific biotinylation of proteins using in vitor, in vivo and cellfree systems for teh purpose of fabricating functional prtoein arrays. Biotinylation of recombinant proteins relies on the chemoselective reaction between cystein-biotin and a reactive thioester group at the C temrinus of a protien generated via intein-mediated cleavage.
Yao “Intein-mediated biotinylation of proteins and its application in a protein microarray” J. Am Chem. Soc. 2002, 124, (2002) discloses an intein-mediated expression system to express, purify and site-specifically biotinylate prtoeins, followed by immobilization onto avidin-functionalized glass slides. Three model proteins with an intein tag (intein fused to chitin binding domain) at their C-termini) were purified and biotinylated in a single step, by first loading the crude cell lystate onto a column packed with chitin beads adn then flushing the column with biotinylated cysteine. Further improvement may be made by using streptavidin as the immobilization agent on the slide, in place of avidin, which is a glycoprotein and known to have higher nonspecific binding characteristics.
Commercially availabe Resins
Thermo Scientific SulfoLink Cupoling Resin: is a porous, crosslinked, 6% beaded agarose that has been activated with iodoacetyle groups for covalent immobilization of cystein-peptides and other sulfhydryl molecules. Iodoacetyl groups react specfically with sulhydryls to form irreversible thioether bonds. The resin has been used for example to immoblilize antibody binding prtoeins such as Protein G onto a resin. Kossiakoff (US 2018/0044385