Page 118 - Tyrosine-Based Bioconjugations - Jorick Bruins
P. 118

Chapter 7
1.0 mg/mL mTyr and 5 eq. of BCN-bearing tag is sufficient, but increasing antibody and BCN-tag concentration may be attempted depending on the protein of interest.
Co-solvents: The majority of the BCN- and cpTCO-probes applied in this thesis are insoluble in water and therefore require a co-solvent such as DMSO or DMF to maintain a homogenous solution. MeOH is an option, but is more volatile and therefore concentrations of the probe stocks may be more variable. Of note, mTyr activity is reduced at increased DMSO concentration, with about 75% activity at 10% (v/v) DMSO and 40% activity at 20% (v/v) DMSO.8
Tyrosinases and other oxidizing reagents: In this thesis, only mushroom tyrosinase (mTyr) was used for the oxidation of tyrosine due to its commercial availability. Smaller tyrosinases such as monomeric mTyr9 or tyrosinase from Burkholderia thailandensis (BtTyr), a faster tyrosinase capable of catalysis at lower pH,10 can be considered. Furthermore, in the unique situation where there is only a single tyrosine present on the protein, Fremy’s salt can also be considered.11
Label size and type: As shown in chapters 2–5, BCN, TCO, and cpTCO are all capable of performing SPOCQ, yielding conjugates with >95% conversion. In chapter 5, we also showed that direct conjugation of larger molecules such as proteins via SPOCQ is unfavorable. Generally, BCN is the optimal reagent for SPOCQ, although bigger probes (>5 kDa) should ideally be conjugated via two-step conjugation (chapter 5).
Tyrosine tag size and location: Both C-terminal and N-terminal tyrosine tags are eligible for conjugation by SPOCQ. When expressing an N-terminal G4Y-tag, an additional amino acid (i.e. methionine) must be engineered to the very N-terminus to prevent intermolecular Michael addition when the quinone generated, otherwise leading into the formation of dopachrome compounds.12 In many cases, expression of a G4Y-tag (C-terminus) or MYG4-tag (N-terminus) is enough for rapid and selective oxidation by mushroom tyrosinase. To improve reaction rate, one or two G4S-spacers can be inserted to increase accessibility of the tyrosine residue (chapter 5). Finally, expressing solvent-exposed tyrosine residues in non-terminal positions has been achieved, but application of this method would require careful engineering depending on the protein of interest.
Future Perspectives
We have shown the capability of SPOCQ for protein labeling, and shown its orthogonality to other, established protein conjugation methods. The unique qualities of SPOCQ are the rapid reaction rate under mild conditions, whilst employing an enzyme that can be extracted from the common mushroom.13 Many improvements may still be made to the procedure, such as the use of bead-immobilized tyrosinases for easy workup.14 Faster oxidation of the tyrosine residues can be achieved by more active enzymes such as tyrosinase from Burkholderia thailandensis (BtTYR),10 potentially capable of full conversion within minutes.
116


























































































   116   117   118   119   120