RGD Reference Report - In Vitro Amidating Processing of Products Expressed by Gene Engineering. - Rat Genome Database

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In Vitro Amidating Processing of Products Expressed by Gene Engineering.

Authors: Yang, YH  Chen, H  Yu, C  Zeng, R  Cui, DF  Wu, XF  Li, BL 
Citation: Yang YH, etal., Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2000;32(3):312-315.
RGD ID: 6483570
Pubmed: PMID:12075461   (View Abstract at PubMed)

To set up an in vitro amidating system, a recombinant human calcitonin with a glycine at C termial (mhCT-Gly) was used as the amidating substrate of recombinant rat peptidylglycine alpha-amidating monooxygenase (rPAM). First, the mhCT-Gly gene was synthesized and cloned into a fusion expression vector to get an expression plasmid pGEXCT. The GST-fused mhCT-Gly was highly expressed in E.coli BL21(DE3) harboring the pGEXCT, and was purified rapidly by affinity chromatography. Second, using the method of ultrafiltration, the rPAM was prepared from the supernatant of cultured transfectant CHO cells which express rPAM stably. Finally, the in vitro amidating experiments were carried out using GST-mhCT-Gly as substrate and the prepared rPAM. The results of dot blot with the specific antibody and of mass spectrum assay indicated that amidating product GST-hCT-NH(2) could be easily detected. This study provides a useful method for the amidation of recombinant products in vitro.

Gene Ontology Annotations    Click to see Annotation Detail View

Biological Process
TermQualifierEvidenceWithReferenceNotesSourceOriginal Reference(s)
protein amidation  IDA 6483570 RGD 

Objects Annotated

Genes (Rattus norvegicus)
Pam  (peptidylglycine alpha-amidating monooxygenase)


Additional Information