[Construction of phage-displayed library by DNA shuffling and the screening of potent hirudin variants].
OBJECTIVE
To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method.


METHODS
PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin.


RESULTS
After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained.


CONCLUSIONS
Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.
Long
Yin
Y
Department of Traditional Chinese Medicine, Fourth Military Medical University, Xi'an 710032, China. longyin@fmmu.edu.cn
Liu
Jia-yun
JY
Liu
Li
L
Wang
Zong-ren
ZR
D000818:Animals; D000915:Antibody Affinity; D000991:Antithrombins / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D032621:DNA Shuffling / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015723:Gene Library; D015498:HIV-2 / Q000737:chemistry; D006320:Hearing Tests; D006629:Hirudins / Q000737:chemistry / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*
D000991:Antithrombins; D017931:DNA Primers; D006629:Hirudins; D019151:Peptide Library; D010455:Peptides