Misfolding-assisted selection of stable protein variants using phage displays.
We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of alpha1-antitrypsin (alpha1AT) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at 37 degrees C). Once the alpha1AT moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint (Cm) of the alpha1AT variant, around 20% of the phage retained binding affinity to anti-alpha1AT antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the alpha1AT variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.
Shin
Jong-Shik
JS
Functional Proteomics Center, Korea Institute of Science and Technology, Hawolgok-dong, Seoul 136-791, Korea.
Ryu
Seung-Hyun
SH
Lee
Cheolju
C
Yu
Myeong-Hee
MH
D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin