RT Journal Article
SR Electronic
T1 The identification of an anti-thrombin molecule via the screening of semi-random DNA libraries
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 282806
DO 10.1101/282806
A1 Fan Feng
A1 Zijin Li
A1 Zhenlang Chen
A1 Le Wang
A1 Jie Huang
A1 Yulin Wan
A1 Yunfan Shi
A1 Qiuyun Liu
YR 2018
UL http://biorxiv.org/content/early/2018/09/06/282806.abstract
AB Thrombosis remains one of the leading causes of mortality and morbidity in the world. Thrombin is a key enzyme involved in the blood clotting processes, which can be intervened by low concentrations of Hirudin. The C-terminal dodecapeptide of Hirudin was capable of inhibiting thrombosis. This peptide has been partially randomized in this report, and the coding sequences have been expressed in yeast as chimerical peptides for secretion into the culture media. Two other semi-random modules have been processed likewise. The supernatant was subsequently tested for anti-thrombin activities. DNA sequencing indicated that the putative positive clone encoded a single serine residue followed by a stop codon. The Ninhydrin assay of the culture supernatant of the positive clone indicated a high content of amino acid. Electrospray Mass Spectrometry showed a distinct peak at 430.5 when the expression products from Pichia pastoris were examined, suggesting that the compound may be a dimannosylated serine, as yeast possesses glycosylation at serine residues. The observed effects of α-Mannosidase treatments on the function of yeast induction products are consistent with this assumption. Partial randomization of peptides and proteins may accelerate directed evolution, yielding unprecedented number of variants for functional interrogation and drug development.