Physical and chemical characters of recombinant human nucleoside diphosphate kinase A.
- Author:
Sheng XIONG
1
;
Chui-Wen QIAN
;
Li HUANG
;
Yi-Fei WANG
;
Mei-Ying ZHANG
;
Jiu-Xiang LI
;
Jiu-Feng YAN
;
Xiao-Ning WANG
;
Xiao-Wei ZHANG
;
Zhi-Gang BI
Author Information
1. Biomedical Research and Development Center, Jinan University, Guangzhou 510630, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Humans;
Molecular Sequence Data;
Molecular Weight;
NM23 Nucleoside Diphosphate Kinases;
chemistry;
isolation & purification;
metabolism;
Recombinant Proteins;
chemistry;
isolation & purification;
Scattering, Radiation;
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- From:
Chinese Journal of Biotechnology
2004;20(1):85-89
- CountryChina
- Language:Chinese
-
Abstract:
To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) and determine its physical and chemical characters, recombinant NDPK-A producing E. coli was cultured in 80L fermentor under high cell density culture (HCDC) conditions. The harvested cells were treated with high pressure to break the cell up, tangential-flow microfiltration to remove the bacteria debris and ultrafiltration to concentrate the filtered solution containing target protein. The crude NDPK-A was purified by ion exchange chromatography with DEAE Sepharose Fast Flow, affinity chromatography with Cibarcron Blue 3GA Sepharose CL-4B and gel filtration with Sephadex G-100. The purity of rhNDPK-A was analyzed with SDS-PAGE and RP-HPLC. The Enzymatic activity was determined with RP-HPLC. The molecular weight (MW) was measured with matrix assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS). The N-terminal residue was sequenced with Edman method. The apparent molecular weight of rhNDPK-A in solution was determined with multiangle laser light-scattering method (MALS). It was found that the purity of rhNDPK-A was 97.3% with SDS-PAGE method and 99.2% with RP-HPLC method. The specific enzymatic activity was (900 +/- 100) u/mg. The molecular weight was 17017, which was 132 less than the calculated value according to the amino acid sequence of NDPK-A. The sequencing result of rhNDPK-A revealed that its N-terminal residue was Ala, which was the second residue on N-terminal of native NDPK-A. The calculated MW of N-terminal deleted rhNDPK-A was 17017, exactly equal to the experimental value. The result of apparent MW determination revealed that rhNDPK-A formed homohexamer in solution with a MW of 102kD. These results suggested that rhNDPK-A possessed character identical to its native counterpart of assembling into hexamer. Confirming the identity of rhNDPK-A to its native counterpart provided a good foundation for drug development and mechanism study of NDPK-A.
