Preliminary Study On The Construction, Expression And Biochemical Characteristics Of A Human Alpha-galactosidase A Mutant

Fabry disease is a rare X- inked inheritance of glyco-sphingoside metabolism disorders. Gene mutation of theα-galactosidase A (GLA) ,which located in Xq22,leads to absence or reduction of GLA activity in lysosomes and results in accumulation of globotriaosylceramide(Gb3) and sphingolipid in cells,tissue and organ.Accumulation of Gb3 and sphingolipid will cause blood circulation problem and further lead to heart and kidney failure, nervous system disorders and death.The incidence of Fabry disease is approximately 1 in 40 000 to 1 in 117 000.The life expectancy of untreated patients will be shortened and most of patient will die at about 40.Enzyme replacement therapy is the most efficient approach in Fabry disease treatment. Sufficient and high activity GLA is the fundamental prerequisite for enzyme replacement therapy. GLA is a glycoprotein, and glycosylation is essential for its function. CHO is the most common used host cell for recombinat glycoprotein expression, but the problem of it lies in its low expression level of recombinat protein.It is reported that gene mutation in the nucleotide sequence of 5'untranslated or coding regions could improve the translational efficiency of the mRNA.Based on the above information, the aim of this project is to improve recombinant GLA expression efficiency in mammalian cells. At first, wild-type and mutant-type GLA were amplified and subcloned into eukaryotic expression vectors.Then the recombinant vectors were transfected into mammalian cells. The monoclonal cells which highly express GLA were screened.Secondly, the cell culture and recombinant protein expression conditions were optimized and the selected cells that highly express GLA were cultured at large-scale. Thirdly, the methods of separation and purification were established to obtain high purity GLA. Finally, the purified samples were subjected to pilot basic biochemical analysis. Here is the mian results of this study.1. Construct recombinant eukaryotic expression vectors The GLA cDNA was cloned from SMMC-7721 cells by RT-PCR.Then primers were designed to specifically amplify the wild-type GLA and mutant GLA ( cytosine, the first base after initiation codon was substituted by guanine).The amplified genes were force-subcloned into pcDNA3.1/myc-His A plasmid containg 6×His tag which facilitate the subsequent purification.The wild-type and mutant-type recombinant plasmids were confirmed by enzyme digestion as well as DNA sequencing, and named as pcDNA3.1/myc-His A-GLA and pcDNA3.1/myc-His A-mutation-GLA ,respectively.2. Screen positive clones and optimize induction conditionsThe recombinant plasmids were transfected into CHO cells and HEK293 cells. After the transfected cells were screened with G418 sulfate for about two weeks, four kinds of monoclonal cells were obtained. After GLA activity detection, the clones with the highest GLA expression level in each group were selected.The CHO cells transfected with wild-type and mutant-type GLA gene were named as CHO(GLA) and CHO(m-GLA). The HEK293cells transfected with wild-type and mutant-type GLA gene were named as HEK293(GLA) and CHO(m-GLA).The cells were grown at identical conditions, and induced with sodium butyrate for 72h. GLA detection results showed that the protein activities in the supernatant of CHO(GLA),CHO(m-GLA),HEK293(GLA) and HEK293(m-GLA) were 8.3×10⁶,14.2×10⁶,0.5×10⁶ and 1.4×10⁶U/L, respectively. It was suggested that for the same host cell, GLA activity of the transfectant with mutant-type GLA plasmid was significantly higher than that with wild-type GLA plasmid.What's more, the scrected GLA activity of CHO cells transfected with mutant-type GLA plasmid was obviously higher than that of HEK293 cells transfected with the same plasmid.Then the cell culture and induction conditions of CHO(GLA) and CHO(m-GLA) were optimized.The results indicated that optimal cellular planting density was about 0.5×10⁶cells/well in the 6-well plate. If the cells density was too high,the nutrition and culture space would be absence.On the other hand,if the density was too low,the protein expression level would be relatively low. The GLA expression level increased with the increasing dose of sodium butyrate,and reached to highest level when the sodium butyrate concentration over 10mmol/L. As for the induction time, the GLA expression increased with the prolonged induced time,and it reached a plateau at about 60h after induction.If the induced time was too long, the cells would die and the secreted protein be inactivated. So we choosed 60 hours as the best induction time. 3. Identification of expression productsThe two of CHO(GLA) and CHO(m-GLA) with high protein activity were cultured at large-scale,and induced by 10mmol/L sodium butyrate for 60 hours.The supernatants were collected and purified with HisTrap^TM FF column. Purified samples were analyzed by SDS-PAGE,and for each sample, there was a clear and single band near the 50kd, consistent with the size of GLA ,indicating that it would be recombinant GLA. Gray scanning analysis results showed that the purity of wile-type and mutant-type GLA were 95.2% and 95.8% respectively, indicating that recombinant GLA with high purity were obtained. The purified samples were then identified by Western blot with anti-GLA and anti-6×His antibodies. We found there were clear bands in the same location near 50kd, and further indicated that the purified protein samples were the target protein(GLA). The specific activity of wild-type GLA and mutant-type GLA was 0.0029 and 0.6935×10⁶U/mg before purification. The specific activity of purified wild-type GLA(0.69×10^<sup>6U/mg) was equivalent with commercialized GLA(0.77×10⁶U/mg).However the specific activity(1.78×10⁶U/mg) of purified mutant-type GLA was 2.3 times than commercialization GLA,suggesting the specific activity of purified mutant-type GLA was 2³ times than wild-type GLA. Because the specific activity of unpurified mutant-type GLA was 10-flod of that of unpurified wild-type GLA, the results indicatied mutation in GLA gene could significantly increase the GLA protein activity and improve the translational effciency of GLA mRNA.4. Study on biochemical properties of GLAWith the purified samples, the relative molecular weight, optimal pH, optimal temperature and enzyme kinetic parameters( Km and Vmax) were analysized. Mass spectrometry analysis results showed that the molecular weight of wild-type and mutant GLA was 54.9×10³and 54.8×10³ respectively. Their optimal pH was around 4.6, similar to commercialized GLA . The specific activity of both wild-type and mutant-type GLA gradually increased as the temperature increased, and reached maximum point at 37℃, which was also similar to commercialized GLA.But at low temperature, the enzyme activity of commercialized GLA was a little higher than that of recombinant proteins. The Michaelis constant(Km) and maximum reaction rate study showed, the Km and Vmax of the wild-type GLA were 2.39μmol/L and 0.0034μmol/min repectively, as for mutant GLA and commercialized GLA, the results were 1.27μmol/L , 0.0036μmol/min and 3.45μmol/L, 0.0045μmol/min, respectively. The results indicated that the substrate affinity of mutant-type GLA was higher than other proteins,and the substrate affinity of commercialized GLA was the lowest.This may be the reason why mutant-type GLA showed higher GLA activity than wild-type GLA or commercialized GLA.In summary, the above results suggested that the recombinant plasmid containing mutant-type GLA gene was contructed by site-directed mutagenesis and transfected into CHO cells and HEK293 cells. The monoclonal cells expressing high activity of GLA were selected, and the GLA expression level of CHO(m-GLA) which transfected mutant-type GLA plasmids was highest in four transfectants. The cell culture and inducation of selected transfectant were optimized and the cells were cultured at large-scale. The GLA activity in the supernatant of CHO(m-GLA) cells was about 10-flod of that of CHO(GLA) cells.The recombinant proteins were purified culture and subjected to SDS-PAGE and Western blot. The results indicated that high purity GLA protein be obtained by only one-step purification. Further biochemical analysis of purified samples manifested there was not significant change in the basic charaters of GLA protein after gene mutation, but the activity and substrate affinity significantly increased.It was infered gene mutation could not only improve the translational effciency of GLA mRNA, but also change the space structure and increase the GLA activity. In short, we have obtained a CHO transfectant which expressed high level of mutant-type GLA, and recombinant GLA of high-purity, high activity was obtained .We hope that recombinant GLA can be be used for Fabry disease treatment in the near furture.