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[Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein].
Nan Fang Yi Ke Da Xue Xue Bao. 2006 Sep; 26(9):1319-25.NF

Abstract

OBJECTIVE

To construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT.

METHODS

Using pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro.

RESULTS

Sequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g.

CONCLUSION

The prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress.

Authors+Show Affiliations

Institute of Clinical Medicine, People's Hospital, Yunyang Medical College, Shiyan 442000, China. sandy530530@hotmail.comNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

chi

PubMed ID

16982446

Citation

Yao, Ling-ling, et al. "[Construction of Prokaryotic Expression Plasmid pET15b-PEP-1-CAT and Expression and Purification of PEP-1-CAT Fusion Protein]." Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, vol. 26, no. 9, 2006, pp. 1319-25.
Yao LL, Wang JN, Huang YZ, et al. [Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein]. Nan Fang Yi Ke Da Xue Xue Bao. 2006;26(9):1319-25.
Yao, L. L., Wang, J. N., Huang, Y. Z., & Guo, L. Y. (2006). [Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein]. Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, 26(9), 1319-25.
Yao LL, et al. [Construction of Prokaryotic Expression Plasmid pET15b-PEP-1-CAT and Expression and Purification of PEP-1-CAT Fusion Protein]. Nan Fang Yi Ke Da Xue Xue Bao. 2006;26(9):1319-25. PubMed PMID: 16982446.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Construction of prokaryotic expression plasmid pET15b-PEP-1-CAT and expression and purification of PEP-1-CAT fusion protein]. AU - Yao,Ling-ling, AU - Wang,Jia-ning, AU - Huang,Yong-zhang, AU - Guo,Ling-yun, PY - 2006/9/20/pubmed PY - 2008/5/31/medline PY - 2006/9/20/entrez SP - 1319 EP - 25 JF - Nan fang yi ke da xue xue bao = Journal of Southern Medical University JO - Nan Fang Yi Ke Da Xue Xue Bao VL - 26 IS - 9 N2 - OBJECTIVE: To construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT. METHODS: Using pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro. RESULTS: Sequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g. CONCLUSION: The prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress. SN - 1673-4254 UR - https://www.unboundmedicine.com/medline/citation/16982446/[Construction_of_prokaryotic_expression_plasmid_pET15b_PEP_1_CAT_and_expression_and_purification_of_PEP_1_CAT_fusion_protein]_ DB - PRIME DP - Unbound Medicine ER -