An evolved Mxe GyrA intein for enhanced production of fusion proteins.
Expressing antibodies as fusions to the non-self-cleaving Mxe GyrA intein enables site-specific, carboxy-terminal chemical modification of the antibodies by expressed protein ligation (EPL). Bacterial antibody-intein fusion protein expression platforms typically yield insoluble inclusion bodies that require refolding to obtain active antibody-intein fusion proteins. Previously, we demonstrated that it was possible to employ yeast surface display to express properly folded single-chain antibody (scFv)-intein fusions, therefore permitting the direct small-scale chemical functionalization of scFvs. Here, directed evolution of the Mxe GyrA intein was performed to improve both the display and secretion levels of scFv-intein fusion proteins from yeast. The engineered intein was shown to increase the yeast display levels of eight different scFvs by up to 3-fold. Additionally, scFv- and green fluorescent protein (GFP)-intein fusion proteins can be secreted from yeast, and while fusion of the scFvs to the wild-type intein resulted in low expression levels, the engineered intein increased scFv-intein production levels by up to 30-fold. The secreted scFv- and GFP-intein fusion proteins retained their respective binding and fluorescent activities, and upon intein release, EPL resulted in carboxy-terminal azide functionalization of the target proteins. The azide-functionalized scFvs and GFP were subsequently employed in a copper-free, strain-promoted click reaction to site-specifically immobilize the proteins on surfaces, and it was demonstrated that the functionalized, immobilized scFvs retained their antigen binding specificity. Taken together, the evolved yeast intein platform provides a robust alternative to bacterial intein expression systems.
Dept. of Chemical and Biological Engineering, University of Wisconsin-Madison , 1415 Engineering Drive, Madison, Wisconsin 53706, United States.
Carrie J
CJ
Marshall
Vanessa A
VA
Grosskopf
Taylor J
TJ
Moehling
Benjamin J
BJ
Tillotson
Gregory J
GJ
Wiepz
Nicholas L
NL
Abbott
Ronald T
RT
Raines
Eric V
EV
Shusta
D057930:Click Chemistry; D027081:DNA Gyrase / Q000737:chemistry* / Q000378:metabolism; D019020:Directed Molecular Evolution; D047668:Inteins / Q000502:physiology*; D008958:Models, Molecular; D015394:Molecular Structure; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D054852:Small Molecule Libraries
D011994:Recombinant Proteins; D054852:Small Molecule Libraries; D027081:DNA Gyrase