EKSPRESI GEN PENYANDI b-XILOSIDASE DALAM SISTEM pHIS1525/ Bacillus megaterium MS941

The aim of this research was to express the β-xylosidase gene in the pHIS1525 or Bacillus megaterium MS941 system. The xyl gene was amplified from pTP510 and cloned into pHIS1525 in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant B. megaterium MS941 on solid LB medium containing tetracycline (10 μg/ml). The expression of β-xylosidase was assayed using 0.2 percent methylumbelliferyl-β-D-xyloside (MUX) and the proteins were analyzed by SDS-PAGE method. The b-xilosidase activity was determined toward p-nitrophenyl-b-Dxylopyranoside (pNPX) as a substrate and p-nitrofenol releasing was measured by UV/Vis spectrophotometer at λ 405 nm. This research showed that recombinant B. megaterium MS941 expressed the β-xylosidase gene (xyl) and secreted it into the culture medium. The SDS-PAGE analysis of extracellular protein (culture medium) showed a 60,0 kD protein band. The recombinant Bacillus megaterium MS941 expressed and secreted the β-xilosidase into culture medium 5 hours after adding 5 percent xylose. The b-xylosidase activity was 0.441 unit/ml toward pNPX as a substrate.

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EXPRESSION OF b-XYLOSIDASE ENCODING GENE IN PHIS/ Bacillus megaterium MS SYSTEM

Indonesian Journal of Tropical and Infectious Disease ◽

10.20473/ijtid.v2i1.185 ◽

2015 ◽

Vol 2 (1) ◽

pp. 25 ◽

Cited By ~ 2

Author(s):

Sri Sumarsih

Keyword(s):

Culture Medium ◽

Culture Supernatant ◽

Specific Activity ◽

Agar Medium ◽

Nitrilotriacetic Acid ◽

Protoplast Transformation ◽

E Coli ◽

Recombinant Cells ◽

Relative Molecular Weight ◽

Encoding Gene

b-Xylosidase encoding gene from G. thermoleovorans IT-08 had been expressed in the pHIS1525/ B. megaterium MS941 system. The b-xylosidase gene (xyl) was inserted into plasmid pHIS1525 and propagated in E. coli DH10b. The recombinant plasmid was transformed into B. megaterium MS941 by protoplast transformation. Transformants were selected by growing the recombinant cells on solid LB medium containing tetracycline (10 µg/ ml). The expression of the b-xylosidase gene was assayed by overlaid the recombinant B. megaterium MS941 cell with agar medium containing 0.2% ethylumbelliferyl-b-D-xyloside (MUX). This research showed that the b-xylosidase gene was succesfully sub-cloned in pHIS1525 system and expressed by the recombinant B. megaterium MS941. Theaddition of 0.5% xylose into the culture medium could increase the activity of recombinantactivity of recombinant of recombinantb-xylosidase by 2.74 fold. The recombinant B. megaterium MS941 secreted 75.56% of the expressed b-xylosidase into culture medium. The crude extract b-xylosidase showed the optimum activity at 50° C and pH 6. The recombinant b-xylosidase was purified from culture supernatant by affinity chromatographic method using agarose containing Ni-NTA (Nickel-Nitrilotriacetic acid). The pure b-xylosidase showed a specific activity of 10.06 Unit/mg protein and relative molecular weight ± 58 kDa.

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Expression of Recombinant Fusion Protein of Newcastle Disease Virus from Escherichia coli Plasmid Clone C-2a by In-vitro Cell-free Protein Expression System

BIO Web of Conferences ◽

10.1051/bioconf/20202004004 ◽

2020 ◽

Vol 20 ◽

pp. 04004

Author(s):

Ahmad Pandu Satria Wiratama ◽

Aris Haryanto

Keyword(s):

Protein Expression ◽

Newcastle Disease ◽

Recombinant Plasmid ◽

Expression System ◽

Free Protein ◽

F Protein ◽

E Coli ◽

Protein Expression System ◽

Sds Page

Newcastle Disease Virus (NDV) is an infectious disease that infect many kinds of wild and domesticated birds. Infection of NDV become a massive problem for poultry industry around the world especially in Indonesia. Vaccination is an effort to prevent the infection of NDV in poultry. NDV vaccine that used in Indonesia is a conventional life vaccine from LaSota and B1 strains. These type of vaccine is 21%-23% genetically distinct with the virus that spread in the environment. The antibody protection provided by the vaccine is not effective. Therefore, vaccination with new local NDV strain is needed to prevent the NDV infection in Indonesia. The previously study research reported that the local isolate of NDV from Kulon Progo, Indonesia has been isolated. Fusion (F) protein encoding gene that has been inserted into pBT7-N-His expression p lasmid which isolated from clone C-2a of E. coli, then it was expressed by the Cell-free protein expression system. The aim of this study was to confirm whether clone C-2a of E.coli carrying a recombinant plasmid pBT7-N-His-Fusion NDV and to express a recombinant F protein of NDV in-vitro from expression plasmid by cell-free protein expression system. This work started by detection of recombinant plasmid pBT7-N-His-Fusion NDV by DNA plasmid extraction followed by agarose gel electrophoresis. The recombinant F protein was in-vitro expressed by cell-free protein expression kit. The expressed F protein of NDV then was visualized by SDS-PAGE and Westernblott to analyse the expression of NDV recombinant F protein. It confirmed that clone C-2a of E. coli contained plasmid pBT7-N-His (4.001 bp) inserted by recombinant F protein of NDV gene (642 bp). The visualisation of expressed recombinant F protein by SDS-PAGE and Westernblott showed the NDV recombinant F protein was a specific protein fragment with molecular weight of 25,6 kDa..

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VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

Journal of Biological Researches ◽

10.23869/bphjbr.11.1.200510 ◽

2005 ◽

Vol 11 (1) ◽

pp. 61-66

Author(s):

Ira Djajanegara ◽

Wayan Artama ◽

Retno Lestari ◽

Sabar Pambudi

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Plasmid ◽

Restriction Site ◽

Standard Procedure ◽

Pcr Amplification ◽

Gene Encoding ◽

E Coli ◽

Gene Coding ◽

Sds Page ◽

Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

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Clonning and expression of recombinant carbapenemase KPC-2 enzyme in E. coli cytoplasm

Science and Technology Development Journal ◽

10.32508/stdj.v19i2.800 ◽

2016 ◽

Vol 19 (2) ◽

pp. 27-37

Author(s):

Phuong Nhat Tran ◽

Phuong Thi Kim Huynh ◽

Trang Thi Phuong Tran ◽

Thuoc Linh Tran ◽

Van Hung Pham

Keyword(s):

Klebsiella Pneumoniae ◽

Clinical Research ◽

Affinity Chromatography ◽

Recombinant Plasmid ◽

Broth Culture ◽

E Coli ◽

Recombinant Vector ◽

Carbapenem Resistant ◽

Sds Page ◽

Encoding Genes

Production of KPC-type carbapenemase is the most common carbapenem resistant mechanism in Klebsiella pneumoniae. The expression level of KPC in these strains is different and is mostly required other mechanisms to reach the higher resistant level such as porin lost or co-expression of extended spectrum β-lactamase (ESBL). To better understand the expression of KPC enzyme, the KPC-2 encoding genes from clinical isolated K. pneumoniae were cloned into pET28a plasmid. The recombinant plasmids containing of kpc-2 gene were subsequently transformed into E. coli OmniMax and were screened in kanamycine added LB media to select E. coli possessing of recombinant plasmid. Carbapenemase activity in the broth culture was checked in LB broth supplemented with 4 µg/mL of ertapenem and the expression induced with IPTG was checked by SDS-PAGE method. The results showed that this recombinant vector was capable of effective expression of KPC-2 protein in E. coli and this strain could be grown in LB broth supplemented with 4 µg/mL of ertapenem. A half of the target protein was soluble in the supernatant however it could be successfully collected from a HistrapHP affinity chromatography column. The result of this report is one of resources for further studies and applications of this KPC-2 protein in clinical research.

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Optimization of Expression, Purification and Secretion of Functional Recombinant Human Growth Hormone in Prokaryotic Hosts Using Modified Staphylococcal Protein A Signal Peptide

10.21203/rs.3.rs-257852/v1 ◽

2021 ◽

Author(s):

Garshasb Rigi ◽

Amin Rostami ◽

Habib Ghomi ◽

Gholamreza Ahmadian ◽

Vasiqe Sadat Mirbagheri ◽

...

Keyword(s):

Growth Hormone ◽

Western Blot ◽

Signal Peptide ◽

Extracellular Space ◽

Culture Medium ◽

Active Form ◽

Protein A ◽

Human Growth ◽

E Coli ◽

Sds Page

Abstract Background: Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the E. coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results: In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm.Conclusions: Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

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Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

BMC Biotechnology ◽

10.1186/s12896-021-00701-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Garshasb Rigi ◽

Amin Rostami ◽

Habib Ghomi ◽

Gholamreza Ahmadian ◽

Vasiqe Sadat Mirbagheri ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Hormone ◽

Signal Peptide ◽

Extracellular Space ◽

Culture Medium ◽

Active Form ◽

Protein A ◽

Human Growth ◽

E Coli ◽

Sds Page

Abstract Background Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. Conclusions Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

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Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli

BioMed Research International ◽

10.1155/2018/3429569 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7

Author(s):

Zhongshan Wang ◽

Xiaokun Xia ◽

Meixian Zhang ◽

Jiawei Fang ◽

Yanqiang Li ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Knockout ◽

Binding Protein ◽

Gel Filtration ◽

Direct Interaction ◽

Protein Band ◽

Sulfur Source ◽

E Coli ◽

Sds Page ◽

Glutathione Binding

Objectives. To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results. The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions. GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.

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Expression of Hepatitis B viral surface antigen (Pre S1) in E. coli

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11i3.2406 ◽

2020 ◽

Vol 11 (3) ◽

pp. 3046-3052

Author(s):

Shahad Basel Ismail ◽

Yaseen Ismael Mamoori ◽

Ibrahim Abdulla Ahmed

Keyword(s):

Molecular Weight ◽

Hepatitis B ◽

Protein Band ◽

Effective Vaccine ◽

Chronic Hepatitis B Infection ◽

Colony Pcr ◽

E Coli ◽

Sds Page ◽

B Virus ◽

Viral Surface

Hepatitis B is the most common liver diseases, which caused by hepatitis B virus (HBV) infection. There are around 257 million people around the world suffer from severe chronic hepatitis B infection. Therefore, it is necessary to develop a vaccine to prevent viral infection. PreS1 is one of the HBV envelope proteins that have been proved to be an effective vaccine. Accordingly, Viral DNA was purified from patients’ serums and amplified by PCR using specific primers. Amplicons of 324 bp bands of PreS1 was observed on gel electrophoresis. The PreS1 was cloned into pTXB21 plasmid to form the recombinant plasmid pTXB1_PreS1 and transformed into DH5α E. coli. Screening of transformants was done using Colony PCR and Sequencing. Alignment of 26 polypeptide sequences showed conservation of this region. The pTXB1_PreS1 was retransformed into T7 Express Competent E. coli and screened using colony PCR. The PreS1 was expressed as a recombinant protein fused to an intein tag with a molecular weight of ~ 39.5 kD. The PreS1 protein was purified by a single affinity chromatography step and after cleaved from intein tag by Dithiothreitol the obtained protein had a molecular weight of ~ 11.5 kD. Only one protein band was observed on the SDS-page gel. The PreS1 protein was successfully cloned and expressed in E. coli, which can be used as a vaccine against HBV.

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Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190416144650 ◽

2019 ◽

Vol 20 (6) ◽

pp. 497-505 ◽

Cited By ~ 1

Author(s):

Abeer M. Abd El-Aziz ◽

Mohamed A. Shaker ◽

Mona I. Shaaban

Keyword(s):

Gold Nanoparticles ◽

Lipolytic Activity ◽

Market Demand ◽

Biotechnological Applications ◽

Lipase Gene ◽

Expression Plasmid ◽

E Coli ◽

Nickel Affinity Chromatography ◽

Recombinant Production ◽

Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

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Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

Journal of Ophthalmology ◽

10.1155/2019/2431481 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 5

Author(s):

Takashi Kanamoto ◽

Takashi Tachibana ◽

Yasushi Kitaoka ◽

Toshio Hisatomi ◽

Yasuhiro Ikeda ◽

...

Keyword(s):

Mass Spectrometry ◽

Ocular Hypertension ◽

Aspartic Acid ◽

Atp Synthase ◽

Wistar Rats ◽

Protein Band ◽

Mass Spectrometry Analysis ◽

Liquid Chromatography Mass Spectrometry ◽

Spectrometry Analysis ◽

Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

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