Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System
Renin is an important honnone in kidney regulating the renin-angiotensin system (RAS); which plays an important role in human blood pressure. Renin is a highly specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate angiotensin I. Recently, renin was found in organs other...
| Main Author: | |
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| Format: | Thesis |
| Language: | English English |
| Published: |
2002
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8486/1/FSMB_2002_23_A.pdf |
| _version_ | 1796967540221542400 |
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| author | Ng, Chyan Leong |
| author_facet | Ng, Chyan Leong |
| author_sort | Ng, Chyan Leong |
| collection | UPM |
| description | Renin is an important honnone in kidney regulating the renin-angiotensin system
(RAS); which plays an important role in human blood pressure. Renin is a highly
specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate
angiotensin I. Recently, renin was found in organs other than the kidney such as
adrenal, ovary, testis, uterus, placenta, anterior pituitary and brain, implicating its
involvement in the regulation of numerous activities. Prorenin is the inactive
precursor of the renin which regulates the blood pressure and electrolyte balance.
Prorenin can be activated in vitro following nonproteolysis and proteolysis. The
isolation of prorenin or renin from organs including kidney is extremely difficult due
to its very low concentration and its instability. Therefore, recombinant protein
technologies are used to produce the recombinant human renin protein.
In this study, the full-length human renin coding gene (REN) was isolated from the
human kidney cDNA library by using the polymerase chain reaction (PCR)
technique. The primers (RF1 & RR1) used were designed based on the human
mRNA renin gene sequence from GenBank [gi |4506474| ref | NM_000537.1|]. The PCR amplified REN gene was cloned into pCR-Blunt cloning vector. Sequencing
was carried out and the result shows 99.9% identical to the published sequence. The
REN gene was cloned into two different E. coli expression vectors, pRSETB and
pGEX4T l , to express the recombinant protein. Construct pRB-R was successfully
expressed in E. coli strains BL2 1-S1 and BL21 (DE3)pLysS with the recombinant
protein corresponding to the expected size -48 kDa. Construct pGT-R was expressed
in BL2 1 (DE3)pLysS with the size -66 kDa. Both recombinant proteins have been
confirmed with western blotting by using monoclonal anti-His antibody
(recombinant protein derived from pRSET vector) and monoclonal anti-GST
antibody (recombinant protein derived from p GEX4Tl vector). The result of the
expression shows that the combination of the expression vector pRSETB and host
BL2 1 (DE3)pLysS gave the highest soluble fraction of recombinant protein. |
| first_indexed | 2024-03-06T07:15:22Z |
| format | Thesis |
| id | upm.eprints-8486 |
| institution | Universiti Putra Malaysia |
| language | English English |
| last_indexed | 2024-03-06T07:15:22Z |
| publishDate | 2002 |
| record_format | dspace |
| spelling | upm.eprints-84862024-01-26T08:56:26Z http://psasir.upm.edu.my/id/eprint/8486/ Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System Ng, Chyan Leong Renin is an important honnone in kidney regulating the renin-angiotensin system (RAS); which plays an important role in human blood pressure. Renin is a highly specific endopeptidase cleaving the Leu-Leu bond in angiotensinogen to generate angiotensin I. Recently, renin was found in organs other than the kidney such as adrenal, ovary, testis, uterus, placenta, anterior pituitary and brain, implicating its involvement in the regulation of numerous activities. Prorenin is the inactive precursor of the renin which regulates the blood pressure and electrolyte balance. Prorenin can be activated in vitro following nonproteolysis and proteolysis. The isolation of prorenin or renin from organs including kidney is extremely difficult due to its very low concentration and its instability. Therefore, recombinant protein technologies are used to produce the recombinant human renin protein. In this study, the full-length human renin coding gene (REN) was isolated from the human kidney cDNA library by using the polymerase chain reaction (PCR) technique. The primers (RF1 & RR1) used were designed based on the human mRNA renin gene sequence from GenBank [gi |4506474| ref | NM_000537.1|]. The PCR amplified REN gene was cloned into pCR-Blunt cloning vector. Sequencing was carried out and the result shows 99.9% identical to the published sequence. The REN gene was cloned into two different E. coli expression vectors, pRSETB and pGEX4T l , to express the recombinant protein. Construct pRB-R was successfully expressed in E. coli strains BL2 1-S1 and BL21 (DE3)pLysS with the recombinant protein corresponding to the expected size -48 kDa. Construct pGT-R was expressed in BL2 1 (DE3)pLysS with the size -66 kDa. Both recombinant proteins have been confirmed with western blotting by using monoclonal anti-His antibody (recombinant protein derived from pRSET vector) and monoclonal anti-GST antibody (recombinant protein derived from p GEX4Tl vector). The result of the expression shows that the combination of the expression vector pRSETB and host BL2 1 (DE3)pLysS gave the highest soluble fraction of recombinant protein. 2002-08 Thesis NonPeerReviewed application/pdf en http://psasir.upm.edu.my/id/eprint/8486/1/FSMB_2002_23_A.pdf Ng, Chyan Leong (2002) Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System. Masters thesis, Universiti Putra Malaysia. Renin. Escherichia coli - Biotechnology. Recombinant proteins. English |
| spellingShingle | Renin. Escherichia coli - Biotechnology. Recombinant proteins. Ng, Chyan Leong Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title | Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title_full | Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title_fullStr | Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title_full_unstemmed | Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title_short | Isolation, Cloning and Expression of Recombinant Human Renin in Escherichia Coli System |
| title_sort | isolation cloning and expression of recombinant human renin in escherichia coli system |
| topic | Renin. Escherichia coli - Biotechnology. Recombinant proteins. |
| url | http://psasir.upm.edu.my/id/eprint/8486/1/FSMB_2002_23_A.pdf |
| work_keys_str_mv | AT ngchyanleong isolationcloningandexpressionofrecombinanthumanrenininescherichiacolisystem |