One-Pot Synthesis of High Molecular Weight Synthetic Heteroprotein Dimers Driven by Charge Complementarity Electrostatic Interactions

Abstract

Despite the importance of protein dimers and dimerization in biology, the formation of protein dimers through synthetic covalent chemistry has not found widespread use. In the case of maleimide-cysteine-based dimerization of proteins, we show here that when the proteins have the same charge, dimerization appears to be inherently difficult with yields around 1% or less, regardless of the nature of the spacer used or whether homo- or heteroprotein dimers are targeted. In contrast, if the proteins have opposing (complementary) charges, the formation of heteroprotein dimers proceeds much more readily, and in the case of one high molecular weight (>80 kDa) synthetic dimer between cytochrome c and bovine serum albumin, a 30% yield of the purified, isolated dimer was achieved. This represents at least a 30-fold increase in yield for protein dimers formed from proteins with complementary charges, compared to when the proteins have the same charge, under otherwise similar conditions. These results illustrate the role of ionic supramolecular interactions in controlling the reactivity of proteins toward bis-functionalized spacers. The strategy here for effective synthetic dimerization of proteins could be very useful for developing novel approaches to study the important role of protein-protein interactions in chemical biology.

Affinity-Based Purification of Polyisocyanopeptide Bioconjugates

“…Compared to biotinPIC without αCD3, these yields are slightly lower, which is a direct result of a less efficient capture of the bioconjugate. This may originate from steric effects resulting from the large antibody or less-favorable charge interactions between the antibody and monoavidin . In the case of unfavorable charge interactions, optimizing the buffer conditions in the capture and/or release step may lead to increased yields in future experiments.…”

PEGylation and Dimerization of Expressed Proteins under Near Equimolar Conditions with Potassium 2-Pyridyl Acyltrifluoroborates

“…Significant advances in this area include the works of Maynard, 89 , 90 Francis, 91 Bertozzi, Nogales and Rabuka, 92 Kluger, 93 and Thordarson. 94 …”

“…The linker should be of sufficient length as to avoid steric and electrostatic clashes. 94 We prepared a 3.4 kDa bis 2-pyridyl PEG KAT 12 in an analogous fashion to our 2-pyridyl mPEG KAT reagents 9a – d ( Scheme 2 ).…”

“…Significant advances in this area include the works of Maynard, 89,90 Francis, 91 Bertozzi, Nogales and Rabuka, 92 Kluger, 93 and Thordarson. 94 Based on the outstanding results of protein PEGylation with KAT reagents under near stoichiometric conditions, we envisioned that a KAT-mediated homodimerization of a protein could be realized, utilizing one equivalent of a bis 2-pyridyl PEG KAT linker and two or more equivalents of protein hydroxylamine. The linker should be of sufficient length as to avoid steric and electrostatic clashes.…”

Homodimeric Protein–Polymer Conjugates via the Tetrazine–trans-Cyclooctene Ligation

“…24 Maleimide-thiol chemistry has also been used with small molecules of varying spacer length to obtain a 30% yield for dimerization of oppositely charged proteins and less than 1% yield for similarly charged proteins, suggesting that charge can facilitate or limit dimer formation. 27 Recent developments in conjugation techniques and linkers may have the potential to produce higher yields of protein dimers. 28–30 Here, we report the use of an extremely rapid bioorthogonal chemistry, specifically the tetrazine ligation, as a method to increase dimerization yield.…”