Conjugation of proteins by installing BIO-orthogonally reactive groups at their N-termini.
N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.
Department of Polymer Science and Chemical Engineering, Pusan National University, Busan, South Korea.
Nagasundarapandian
N
Soundrarajan
Sriram
S
Sokalingam
Govindan
G
Raghunathan
Nediljko
N
Budisa
Hyun-Jong
HJ
Paik
Tae Hyeon
TH
Yoo
Sun-Gu
SG
Lee
D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry; D000480:Alkynes / Q000737:chemistry; D057930:Click Chemistry / Q000379:methods*; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D000480:Alkynes; D011506:Proteins; C506098:azidohomoalanine; C551041:homopropargylglycine; D049452:Green Fluorescent Proteins; D008715:Methionine; D000409:Alanine; D005998:Glycine