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Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris

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@article{IJASEIT912,
   author = {Ratih Asmana Ningrum and Neng Herawati and Andri Wardiana},
   title = {Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris},
   journal = {International Journal on Advanced Science, Engineering and Information Technology},
   volume = {7},
   number = {1},
   year = {2017},
   pages = {8--14},
   keywords = {human interferon alpha-2a; human serum albumin; fusion protein; antiproliverative; Pichia pastoris},
   abstract = {

Human interferon alpha-2a (hIFNα-2a) is a therapeutic protein that used in cancer and hepatitis B/C treatments. One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims to develop recombinant hIFNα-2a fusion protein by using human serum albumin (HSA) to improve its molecular weight. The codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted into pPICZαB expression vector and transformed into Escherichia coli XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast Pichia pastoris GS115 and SMD 1168. The expression analysis showed that the best expression was achieved by protease deficient strain SMD1168. Molecular weight characterization informed that the fusion protein was 85 kDa in size as its theoritical size. Western blotting methods confirmed the identity of fusion protein based on the recognition by anti HSA and anti hIFNα-2 antibodies. This result strongly indicated that the fusion protein was successfully produced in Pichia pastoris. Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60th generations. Preliminary antiproliverative activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form.  

},    issn = {2088-5334},    publisher = {INSIGHT - Indonesian Society for Knowledge and Human Development},    url = {http://ijaseit.insightsociety.org/index.php?option=com_content&view=article&id=9&Itemid=1&article_id=912},    doi = {10.18517/ijaseit.7.1.912} }

EndNote

%A Ningrum, Ratih Asmana
%A Herawati, Neng
%A Wardiana, Andri
%D 2017
%T Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris
%B 2017
%9 human interferon alpha-2a; human serum albumin; fusion protein; antiproliverative; Pichia pastoris
%! Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris
%K human interferon alpha-2a; human serum albumin; fusion protein; antiproliverative; Pichia pastoris
%X 

Human interferon alpha-2a (hIFNα-2a) is a therapeutic protein that used in cancer and hepatitis B/C treatments. One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims to develop recombinant hIFNα-2a fusion protein by using human serum albumin (HSA) to improve its molecular weight. The codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted into pPICZαB expression vector and transformed into Escherichia coli XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast Pichia pastoris GS115 and SMD 1168. The expression analysis showed that the best expression was achieved by protease deficient strain SMD1168. Molecular weight characterization informed that the fusion protein was 85 kDa in size as its theoritical size. Western blotting methods confirmed the identity of fusion protein based on the recognition by anti HSA and anti hIFNα-2 antibodies. This result strongly indicated that the fusion protein was successfully produced in Pichia pastoris. Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60th generations. Preliminary antiproliverative activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form.  

%U http://ijaseit.insightsociety.org/index.php?option=com_content&view=article&id=9&Itemid=1&article_id=912 %R doi:10.18517/ijaseit.7.1.912 %J International Journal on Advanced Science, Engineering and Information Technology %V 7 %N 1 %@ 2088-5334

IEEE

Ratih Asmana Ningrum,Neng Herawati and Andri Wardiana,"Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris," International Journal on Advanced Science, Engineering and Information Technology, vol. 7, no. 1, pp. 8-14, 2017. [Online]. Available: http://dx.doi.org/10.18517/ijaseit.7.1.912.

RefMan/ProCite (RIS)

TY  - JOUR
AU  - Ningrum, Ratih Asmana
AU  - Herawati, Neng
AU  - Wardiana, Andri
PY  - 2017
TI  - Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris
JF  - International Journal on Advanced Science, Engineering and Information Technology; Vol. 7 (2017) No. 1
Y2  - 2017
SP  - 8
EP  - 14
SN  - 2088-5334
PB  - INSIGHT - Indonesian Society for Knowledge and Human Development
KW  - human interferon alpha-2a; human serum albumin; fusion protein; antiproliverative; Pichia pastoris
N2  - 

Human interferon alpha-2a (hIFNα-2a) is a therapeutic protein that used in cancer and hepatitis B/C treatments. One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims to develop recombinant hIFNα-2a fusion protein by using human serum albumin (HSA) to improve its molecular weight. The codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted into pPICZαB expression vector and transformed into Escherichia coli XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast Pichia pastoris GS115 and SMD 1168. The expression analysis showed that the best expression was achieved by protease deficient strain SMD1168. Molecular weight characterization informed that the fusion protein was 85 kDa in size as its theoritical size. Western blotting methods confirmed the identity of fusion protein based on the recognition by anti HSA and anti hIFNα-2 antibodies. This result strongly indicated that the fusion protein was successfully produced in Pichia pastoris. Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60th generations. Preliminary antiproliverative activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form.  

UR - http://ijaseit.insightsociety.org/index.php?option=com_content&view=article&id=9&Itemid=1&article_id=912 DO - 10.18517/ijaseit.7.1.912

RefWorks

RT Journal Article
ID 912
A1 Ningrum, Ratih Asmana
A1 Herawati, Neng
A1 Wardiana, Andri
T1 Development of Higher Molecular Weight of Recombinant Human Interferon Alpha-2a by Albumin Fusion Technology in Methilotropic Yeast Pichia Pastoris
JF International Journal on Advanced Science, Engineering and Information Technology
VO 7
IS 1
YR 2017
SP 8
OP 14
SN 2088-5334
PB INSIGHT - Indonesian Society for Knowledge and Human Development
K1 human interferon alpha-2a; human serum albumin; fusion protein; antiproliverative; Pichia pastoris
AB 

Human interferon alpha-2a (hIFNα-2a) is a therapeutic protein that used in cancer and hepatitis B/C treatments. One main problem of using hIFNα-2a is the lack of good pharmacokinetic profile due to its low molecular weight. This research aims to develop recombinant hIFNα-2a fusion protein by using human serum albumin (HSA) to improve its molecular weight. The codon optimized open reading frame (ORF) encoding fusion protein constructed synthetically and inserted into pPICZαB expression vector and transformed into Escherichia coli XL1-blue. The characterized recombinant plasmid was linearized and transformed into methilotropic yeast Pichia pastoris GS115 and SMD 1168. The expression analysis showed that the best expression was achieved by protease deficient strain SMD1168. Molecular weight characterization informed that the fusion protein was 85 kDa in size as its theoritical size. Western blotting methods confirmed the identity of fusion protein based on the recognition by anti HSA and anti hIFNα-2 antibodies. This result strongly indicated that the fusion protein was successfully produced in Pichia pastoris. Protein quantification informed that the protein yield was 14 mg/L (OD600=2). Stability expression analysis showed that protein production was stable until 60th generations. Preliminary antiproliverative activity assay demonstrated that the fusion protein had 20% lower activity comparing to non fusion form.  

LK http://ijaseit.insightsociety.org/index.php?option=com_content&view=article&id=9&Itemid=1&article_id=912 DO - 10.18517/ijaseit.7.1.912