Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria
Lactococcus lactis is a promising microbial cell factory for the production of recombinant proteins like Nattokinase (NK), a key bio-therapeutic enzyme for cardiovascular diseases. During expression, cells could exhibit stress response due to the presence of protein aggregates. Currently, limited st...
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| Format: | Final Year Project (FYP) |
| Language: | English |
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2016
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| Online Access: | http://hdl.handle.net/10356/66963 |
| _version_ | 1811686557737811968 |
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| author | Tay, Stephanie Victoria Choo Gek |
| author2 | Dave Ow Siak Wei |
| author_facet | Dave Ow Siak Wei Tay, Stephanie Victoria Choo Gek |
| author_sort | Tay, Stephanie Victoria Choo Gek |
| collection | NTU |
| description | Lactococcus lactis is a promising microbial cell factory for the production of recombinant proteins like Nattokinase (NK), a key bio-therapeutic enzyme for cardiovascular diseases. During expression, cells could exhibit stress response due to the presence of protein aggregates. Currently, limited studies are conducted on co-expressing molecular chaperones to refold protein aggregates in L. lactis. This study aims to investigate whether constitutive co-expression of chaperones facilitate protein folding of NK in L. lactis. Western blot and fibrin assay demonstrated that pNZ8148-NK and pNZ8148-USP45-NK expressed from 100 ng/mL over 10 ng/mL of nisin had higher expression and activity in soluble and secreted fractions while insoluble fractions had no functional activity. Attempts at cloning of constitutive promoters: p59 and pUSP45 and chaperones: ClpX, Dnak, Hsp33 and Trigger Factor from Lactobacillus plantarum using T4 ligase cloning and Gibson assembly led to the successful cloning of constitutive promoters into pORI198 co-expression plasmid but not in pNZ9530. Further cloning optimization is required to clone the chaperones into pORI198 and introduce into pNZ8148-NK-NZ9000 and pNZ8148-USP45-NK-NZ9000 cells to improve NK protein folding. We hope to gain insights on the role of molecular chaperones in facilitating protein folding and improve L. lactis as an expression system for recombinant protein production. |
| first_indexed | 2024-10-01T05:02:19Z |
| format | Final Year Project (FYP) |
| id | ntu-10356/66963 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:02:19Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | ntu-10356/669632023-02-28T18:01:49Z Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria Tay, Stephanie Victoria Choo Gek Dave Ow Siak Wei School of Biological Sciences A*STAR Bioprocessing Technology Institute DRNTU::Science::Biological sciences Lactococcus lactis is a promising microbial cell factory for the production of recombinant proteins like Nattokinase (NK), a key bio-therapeutic enzyme for cardiovascular diseases. During expression, cells could exhibit stress response due to the presence of protein aggregates. Currently, limited studies are conducted on co-expressing molecular chaperones to refold protein aggregates in L. lactis. This study aims to investigate whether constitutive co-expression of chaperones facilitate protein folding of NK in L. lactis. Western blot and fibrin assay demonstrated that pNZ8148-NK and pNZ8148-USP45-NK expressed from 100 ng/mL over 10 ng/mL of nisin had higher expression and activity in soluble and secreted fractions while insoluble fractions had no functional activity. Attempts at cloning of constitutive promoters: p59 and pUSP45 and chaperones: ClpX, Dnak, Hsp33 and Trigger Factor from Lactobacillus plantarum using T4 ligase cloning and Gibson assembly led to the successful cloning of constitutive promoters into pORI198 co-expression plasmid but not in pNZ9530. Further cloning optimization is required to clone the chaperones into pORI198 and introduce into pNZ8148-NK-NZ9000 and pNZ8148-USP45-NK-NZ9000 cells to improve NK protein folding. We hope to gain insights on the role of molecular chaperones in facilitating protein folding and improve L. lactis as an expression system for recombinant protein production. Bachelor of Science in Biological Sciences 2016-05-06T08:12:42Z 2016-05-06T08:12:42Z 2016 Final Year Project (FYP) http://hdl.handle.net/10356/66963 en Nanyang Technological University 41p. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Tay, Stephanie Victoria Choo Gek Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title | Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title_full | Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title_fullStr | Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title_full_unstemmed | Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title_short | Co-expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| title_sort | co expression of protein chaperones for recombinant protein production in lactic acid bacteria |
| topic | DRNTU::Science::Biological sciences |
| url | http://hdl.handle.net/10356/66963 |
| work_keys_str_mv | AT taystephanievictoriachoogek coexpressionofproteinchaperonesforrecombinantproteinproductioninlacticacidbacteria |