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1

Methods of synthesis of modified oligonucleotides and their anlogues-potential inhibitors of gene expression by ANTSENSE strategy

100%

Okruszek A.

Biotechnologia

|

1994

|

issue 4

16-39

EN

A review of current methods employed for the preparation of modified oligodeoksyribonucleotides and their analogues.The most typical analogues are represented including those with preserved phosphate-sugar skeleton and those containing no phosphous in he"internucleotide" bridge and/or no sugar residue.

2

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

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2003

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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

3

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

63%

Sierant M., Okruszek A.

Biotechnologia

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2004

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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.

4

Antisense oligonucleotides against inducible nitric oxide synthase reduce No production in RAW 264.7 cells

45%

Bilecki W., Okruszek A., Kwinkowski M., Sanak M., Przewlocki R.

Biotechnologia

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1996

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issue 4

173-182

EN

Several oligodeoxyribonucleotides in a nuclease-resistant phosphorothioate form targeted to iNOS mRNA were tested in RAW 264,7 cells as potential antisense inhibitors.Antisense S-ODNs inhibited iNOS activity in a time- and dose-depended manner.Application of Lipofectin agent, which itself had no significant effect, greatly increased antisense activity.Decreased levels of the target mRNA, as demonstrated by reverse transcriptase -polymerase chain reaction (RT-PCR), suggest tha RNase H mediated mechanism.

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4 Biotechnologia

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1 Bilecki W.

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1 Sanak M.

Methods of synthesis of modified oligonucleotides and their anlogues-potential inhibitors of gene expression by ANTSENSE strategy

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

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

Antisense oligonucleotides against inducible nitric oxide synthase reduce No production in RAW 264.7 cells