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Recombinant Expression and Purification of wild-type Tomato Thymidine Kinase 1 for Crystallization Experiments

Orozco Rodriguez, Juan Manuel LU (2015) KBKM01 20151
Pure and Applied Biochemistry
Abstract
In the present study, the enzyme thymidine kinase 1 from tomato (ToTK1) was expressed in E.coli and purified to near homogeneity by means of affinity chromatography. However, purified ToTK1 of bacterial origin displayed no substantial catalytic activity. Expression was also attempted
in insect cells using the baculovirus expression vector system (BEVS). Significant TK activities were detected in the crude extracts of insect cells transfected with baculoviruses bearing N- and C-terminal His-tagged ToTK1 constructs. ToTK1 is a protein of interest and medical relevance
because it has recently been identified as a potential candidate for suicide gene therapy for the treatment of malignant gliomas in combination with the nucleoside analogue... (More)
In the present study, the enzyme thymidine kinase 1 from tomato (ToTK1) was expressed in E.coli and purified to near homogeneity by means of affinity chromatography. However, purified ToTK1 of bacterial origin displayed no substantial catalytic activity. Expression was also attempted
in insect cells using the baculovirus expression vector system (BEVS). Significant TK activities were detected in the crude extracts of insect cells transfected with baculoviruses bearing N- and C-terminal His-tagged ToTK1 constructs. ToTK1 is a protein of interest and medical relevance
because it has recently been identified as a potential candidate for suicide gene therapy for the treatment of malignant gliomas in combination with the nucleoside analogue azidothymidine (AZT). The work performed as part of this study represents a major step towards the production of crystallized samples and the elucidation of the 3D structure of the first thymidine kinase of plant origin. (Less)
Popular Abstract
Some types of cancer, brain tumors for example, are very difficult to treat. Many patients die only a few months after being diagnosed and there are very few treatments available. For that reason, the medical community is looking for alternative therapies. One of those therapies is called suicide gene therapy and it consists of combining an enzyme (a protein that carries out a chemical reaction) with certain types of drugs called nucleoside analogs in order to kill cancer cells. The aim of my degree project was to use bacteria and insect cells that have been modified by genetic engineering in order to make them produce an enzyme called ToTK1 (tomato thymidine kinase 1), which is likely candidate to be used in suicide gene therapy.
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author
Orozco Rodriguez, Juan Manuel LU
supervisor
organization
course
KBKM01 20151
year
type
H2 - Master's Degree (Two Years)
subject
keywords
BEVS, E. coli, protein purification, thymidine kinase, protein expression, suicide gene therapy, applied biochemistry, tillämpad biokemi
language
English
id
8054390
date added to LUP
2015-10-16 08:42:33
date last changed
2015-10-16 08:42:33
@misc{8054390,
  abstract     = {In the present study, the enzyme thymidine kinase 1 from tomato (ToTK1) was expressed in E.coli and purified to near homogeneity by means of affinity chromatography. However, purified ToTK1 of bacterial origin displayed no substantial catalytic activity. Expression was also attempted
in insect cells using the baculovirus expression vector system (BEVS). Significant TK activities were detected in the crude extracts of insect cells transfected with baculoviruses bearing N- and C-terminal His-tagged ToTK1 constructs. ToTK1 is a protein of interest and medical relevance
because it has recently been identified as a potential candidate for suicide gene therapy for the treatment of malignant gliomas in combination with the nucleoside analogue azidothymidine (AZT). The work performed as part of this study represents a major step towards the production of crystallized samples and the elucidation of the 3D structure of the first thymidine kinase of plant origin.},
  author       = {Orozco Rodriguez, Juan Manuel},
  keyword      = {BEVS,E. coli,protein purification,thymidine kinase,protein expression,suicide gene therapy,applied biochemistry,tillämpad biokemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Recombinant Expression and Purification of wild-type Tomato Thymidine Kinase 1 for Crystallization Experiments},
  year         = {2015},
}