Chemical Synthesis Of Integral Membrane Proteins

Integral membrane proteins are encoded by a quarter of the genes both in prokaryotic and eukaryotic organisms, based on the analysis of open reading frames. Integral membrane proteins include transporters, linkers, channels, receptors, enzymes, structural membrane-anchoring domains, proteins involved in accumulation and transduction of energy, and proteins responsible for cell adhesion. Membrane proteins and especially multi-membrane spanning proteins are important targets in biotechnological research and drug development. But the traditional recombinant expression strategy has difficulties to generate sufficient amounts of desired proteins with high efficiency. Semi or total chemical synthesis of integral membrane proteins via peptide segment ligation strategies has been proposed as an alternative approach for obtaining integral membrane proteins. Therefore, the development of efficient chemical methods for total or semi synthesis of integral membrane proteins is an important challenge at the interface between chemistry and biology.The recent advances in the chemical synthesis of integral membrane proteins have been reviewed, with particular focus on the methods for difficult peptide synthesis, purification, and enhancement of peptide solubility under the ligation conditions. The applications of these methods to the synthesis of some integral membrane proteins with one or multiple transmembrane domains are described. We believe that the current technical challenge is how to improve the solubility of integral membrane proteins and their fragment peptides.Previous studies have shown that multiply fluorinated alcohols exhibit good ability to dissolve difficult peptide sequences, especially hexafluoro-2-propanol (HFIP). We systematically studied the capability of solvents containing different percentage of HFIP in dissolving transmembrane peptides. Through both HPLC and UV analyses we concluded that60%HFIP/8M Urea constituted a good solvent system. And we demonstrated that native chemical ligation reaction can perform in this optimized solvent system.Finally, we chemical synthesized a membrane protein---inward rectifier potassium ion channel16(Kir5.1) with116amino acids by using Fmoc/tBu/TFA SPPS. A strategy of serine or threonine side-chain Arg-tag was used to enhance the solubility of peptides. This strategy may be a promising strategy for chemical synthesis of other integral membrane proteins.