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Expression level of Ubc9 protein in rat tissues.

100%
Gołębiowski F., Szulc A., Sakowicz M., Szutowicz A., Pawełczyk T.
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2003
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vol. 50
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issue 4
1064-1073
EN
Ubc9 is a homologue of the E2 ubiquitin conjugating enzyme and participates in the covalent linking of SUMO-1 molecule to the target protein. In this report we describe a simple and efficient method for obtaining pure human recombinant Ubc9 protein. The purified Ubc9 retained its native structure and was fully active in an in vitro sumoylation assay with the promyelocytic leukaemia (PML) peptide as a substrate. In order to better understand the physiology of Ubc9 protein we examined its levels in several rat tissues. Immunoblot analyses performed on tissue extracts revealed quantitative and qualitative differences in the expression pattern of Ubc9. The Ubc9 protein was present at a high level in spleen and lung. Moderate level of Ubc9 was detected in kidney and liver. Low amount of Ubc9 was observed in brain, whereas the 18 kDa band of Ubc9 was barely visible or absent in heart and skeletal muscle. In heart and muscle extracts the Ubc9 antibodies recognized a 38 kDa protein band. This band was not visible in extracts of other rat tissues. A comparison of the relative levels of Ubc9 mRNA and protein indicated that the overall expression level of Ubc9 was the highest in spleen and lung. In spleen, lung, kidney, brain, liver and heart there was a good correlation between the 18 kDa protein and Ubc9 mRNA levels. In skeletal muscle the Ubc9 mRNA level was unproportionally high comparing to the level of the 18 kDa protein. The presented data indicate that in the rat the expression of the Ubc9 protein appears to have some degree of tissue specificity.
2
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A systematic investigation of the stability of green fluorescent protein fusion proteins

100%
Janczak M., Bukowski M., Górecki A., Dubin G., Dubin A., Wladyka B.
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2015
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vol. 62
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issue 3
407-411
EN
X-ray crystallography provides important insights into structure-function relationship in biomolecules. However, protein crystals are usually hard to obtain which hinders our understanding of multiple important processes. Crystallization requires large amount of protein sample, whereas recombinant proteins are often unstable or insoluble. Green fluorescent protein (GFP) fusion is one of the approaches to increase protein synthesis, solubility and stability, facilitating crystallization. In this study we analyze the influence of the linker length, composition and the position of GFP relative to the fusion partner on the fusion protein production and stability. To this end, multiple constructs of enzymatically impaired variant of PemKSa toxin from Staphylococcus aureus CH91 fused to GFP were generated. Fusion protein production in Escherichia coli was evaluated. The proteins were purified and their stability tested. PemKSa-α14aa-GFP fusion provided best production and stability. Obtained results demonstrate the importance of optimization of fusion protein construct, including linker selection and the order of fusion partners, in obtaining high quantities of stable protein for crystallization.
3
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Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity.

88%
Sakowicz M., Grdeń M., Pawełczyk T.
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2001
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vol. 48
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issue 3
745-754
EN
In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with β-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney >spleen >lung >heart >brain >muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney >spleen >lung >brain >heart >skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.
4
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Cloning and expression of a new recombinant thrombolytic and anthithrombotic agent - a staphylokinase variant

75%
Kowalski M., Brown G., Bieniasz M., Oszajca K., Chabielska E., Pietras T., Szemraj Z., Makandjou-Ola E., Bartkowiak J., Szemraj J.
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issue 1
41-53
EN
To develop a more potent antithrombin agent with thrombolytic and antiplatelet properties, a new staphylokinase (SAK) variant was constructed. The kringle 2 domain (K2) of tissue type-plasminogen activator (t-PA) containing a fibrin-specific binding site (i), the RGD sequence (Arg-Gly-Asp) for the prevention of platelet aggregation (ii) and the antithrombotic agent - hirulog (iii) was assembled to the C-terminal part of recombinant staphylokinase (r-SAK). cDNA for the hybrid protein SAK-RGD-K2-Hirul was cloned into Pichia pastoris pPIC9K yeast expression vector. The introduction of K2 t-PA, the RGD sequence and hirulog into the C-terminus of r-SAK did not alter the staphylokinase activity. We observed a higher clot lysis potency of SAK-RGD-K2-Hirul as evidenced by a faster and more profound lysis of 125I-labeled human fibrin clots. The potency of thrombin inhibition by the hirulog C-terminal part of the recombinant fusion protein was almost identical to that of r-Hir alone. These results suggest that the SAK-RGD-K2-Hirul construct can be a more potent and faster-acting thrombolytic agent with better antithrombin and antiplatelet properties compared to r-SAK and SAK-RGD-K2-Hir.
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