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| Development of Recombinant Human PepsinogenⅡ Calibrators Derived from Hansenula polymorpha |
| CHEN Dan, GUO Yu-zheng, BAN Jing-yang, LI Lu, WANG Wei-long, LI Ding-feng, LIU Yong |
| Abzymo Biosciences Co. Ltd., Beijing 100176, China |
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Abstract Objective:To efficiently produce the recombinant PGⅡ calibrators for in vitro diagnostic reagents. Methods:The PGⅡ gene designed with the H. polymorpha-prefered codon was cloned into expression vector pRMHP2.1 carrying Zeocin and G418 dual screening markers. After electroporation and rounds of passage and stabilization, the recombinant high-level secretory expression strain 26012/PGⅡwas screened. Furthermore, the recombinant PGⅡ protein was purified using Ni-affinity chromatography from the fed-batch cultures in a 200L fermenter. After determined by a latex-enhanced immunoturbidimetric kit, the purified PGⅡ protein was diluted into serial calibrators with 6 different concentrations and the real-time stability, accelerated stability and on-board stability testing was conducted. Results:The screened recombinant expression strain 26012/PGⅡ, with the PGⅡ gene integrated into the host rDNA site and the gene copy number not less than 40, yield more than 50mg/L PGⅡ of cultures in a secretory form. The purity of the purified recombinant PGⅡ protein was 93.8% using Ni-affinity chromatography from the fed-batch cultures, and the ratio of protein concentration determined using the immunoturbidimetric kit to that using BCA method was about 0.85. After diluted into serial calibrators, the general degradation limit of 10% active content was observed in a1-year real-time stability testing, or a 2-week saccelerated stability testing, or a 2-weekson-board stability testing. Furthermore, it was also shown that the stability of purified recombinant PGⅡ was not inferior to that of commercial PGⅡcalibrators. Conclusions:Recombinant PGⅡ protein secreted from H. polymorpha is suitable for use as the PGⅡ calibrators for in vitro diagnostic reagents.
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Received: 15 January 2016
Published: 25 September 2016
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