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Construction of Human-like Collagen Expression Vector and Its Expression in Pichia Pastoris


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Construction of Human-like Collagen Expression Vector and Its Expression in Pichia Pastoris
GAO Li-hu1YANG Shu-lin1CHU Wei-hua2LI Xin-zhu1NIU Xiao-song1YANG Yan1
1.School of Chemical Engineering,NUST,Nanjing 210094,China;2.School of Life Science and Technology,China Pharmaceutical University,Nanjing 210009,China
human-like collagen polygene Pichia pastoris secretion expression
A human-like collagen monomeric gene(gel) is designed and synthesized according to the unique character of collagenous domain,which is made up of total repeat of "Gly-X-Y" unit.The gel repeats in the same orientation forming six times ploygene,which is inserted into the EcoRI/NotI sites of vector pPIC9K with a HIS4 selectable marker through designed adaptors.After verifying the validity of construction by DNA sequencing,the recombinant vector pPIC9KG6 is electroporated into the Pichia pastoris GS115,and the transformants are screened on YPD medium with gradient concentration of G418 to determine the copy number of the human-like collagen ploygene.The human-like collagen is expressed and secreted with methanol fed-batch inducement by adding per 24 hours for maintaining at 1% concentration.The SDS-PAGE result shows that the expressed human-like collagen is extracellular protein and its apparent molecular weight is 112 ku.


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Last Update: 2008-04-30