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1

Chemically modified siRNA duplexes

100%
Sierant M.
Biotechnologia
|
2010
|
issue 3
146-172
EN
RNA interference (RNAi) is a powerful biological process for a sequence-specific silencing of a gene expression in diverse eukaryotic cells. Discovery of this phenomena had tremendous significance for functional genomics and development of novel gene-specific therapies. The future success of RNAi technology relies on identifying appropriate chemical modifications to improve stability, potency and in vivo cellular delivery of the effector moieties, siRNAs. The presented review attempts to describe some of the biological challenges associated with using synthetic siRNAs to effect RNAi and summarizes the role of the chemical tools in the application of RNAi technology.
2

Inclusion bodies and their application for the production of recombinant proteins in Escherichia coli expression system

63%
Sierant M., Okruszek A.
Biotechnologia
|
2004
|
issue 1
9-19
EN
Over-expression of recombinant proteins in Escherichia coli, the most frequently used prokaryotic expression system, often results in the formation of intracellulary aggregated, insoluble folding intermediates. It is generally thought that protein aggregation is triggered by the failure of polypeptide intermediates to complete folding, leading to self-association. These aggregates are known as inclusion bodies or refractile bodies, since they appear upon microscopic observation as highly refractile areas. The formation of inclusion bodies often increases the yields of recombinant proteins and falicitates their isolation. The aggregated proteins are usually protected from proteases and do not harm host cells. Specific strategies are developed to produce bio-active proteins with the participation of inclusion bodies. These procedures include: 1) isolation and purification of inclusion bodies, 2) solubilization of the protein aggregates, and 3) renaturation of solubilized proteins involving formation of native disulphide bonds.
3

Construction of synthetic gene coding for K2L-tPA ? deletion variant of tissue plasminogen activator. Expression in E. coli and renaturation of recombinant protein

63%
Sierant M., Okruszek A.
Biotechnologia
|
2003
|
issue 4
124-141
EN
2 The human fibrinolytic system comprises an inactive proenzyme ? plasminogen which can be converted to the active form ? plasmin which, in turn, degrades fibrin clots into soluble fibrin degradation products. Tissue plasminogen activator (t-PA) has been identified as a main physiological factor responsible for plasminogen - plasmin conversion. The high fibrin specificity of t-PA, which allows efficient activation on the surface of fibrin clots, has stimulated great interest in its preparation to be used for thrombolytic therapy. Several approaches have been followed to further improve the thrombolytic properties of recombinant t-PA by protein engineering to enhance its plasminogen - activating potency as well as fibrin specificity, and to reduce its plasma clearance. One of the approaches involves the conjugation of its deletion variant, so called K2L-tPA, to various biomolecules. K2L-tPA is a 351-aminoacid C-terminal fragment of human t-PA. The protein is composed of two major domains: Kringle-2 (K2), responsible for fibrin binding, and so-called Light Chain domain (L), containing active centre of the enzyme. In this paper, we describe our efforts on expression of synthetic gene coding for K2L-tPA, and on renaturation and purification of recombinant protein
4

Short interfering RNAs as tools for sequence-specific gene silencing

63%
Sierant M., Nawrot B.
Biotechnologia
|
2003
|
issue 2
84-103
EN
In diverse eukaryotes, dsRNA triggers the destruction of mRNA sharing the same sequence as the dsRNA in the process called RNAi. The guides for sequence-specific degradation of mRNA are 21 nt short interfering RNAs (siRNAs). Synthetic siRNAs can efficiently mediate RNAi, but a drawback of RNAi is its transient nature as a result of the limited availability and stability of synthetic oligonucleotides. Recently, several groups reported the construction of expression plasmid vectors that mediate the production of siRNAs under control of Pol III promoters. These vectors allow the continued expression of siRNAs in the cells resulting in persistent and specific suppression of target genes. The retroviral siRNA expressing system allows for stable inactivation of the genes in primary cells or living organisms.
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