فیملی بزنس میں'' ابہام'' سے پیدا ہونے والے مسائل کا شرعی و تحقیقی جائزہ

ABSTRACTFamily Business is a very important form of business in this era and especially because of this it merely does not matter the business, but more sensitivity is of close relatives and relationships. That is why it has many administrative, Shariah and ethical complications. That is why, this theme has been created as a field of discussion and research. If the issues of Family Business are reviewed, their root is to leave the matters undefined, uncleared and undocumented. Then the solution to all these issues is to overcome their ‘Unclarity’ found in different aspects. To finish the matter, we should clear and correct for example ‘business status’ between father and sons, uncle and nephew or a few brothers. Similarly, in case of death of elders, the inheritance is to be determined. Then that all the matters should be written in a very brief manner and arranged. It is also possible to calculate the income and expenditure account. It is not appropriate that every partner should spend without any speculation. Then one time it becomes difficult to face each other. In this article, we have tried to review all the dimensions where there may be more problems due to ‘unclearity’ in mutual business dealings. In this context, the first business status of family members has been explained. Then mentioned the problems raised after the death of the family leader. Later on, more aspects of the problem are presented by presenting some other observation examples on this subject.

Molecular Characterization of Human Recombinant Interleukin-1Ra Mutants

Aim of the present study is the expression of biological active interleukin 1receptor antagonist (IL-1Ra) and its mutants in prokaryotic as well as in eukaryotic expression system. cDNA was isolated from human placenta and gallbladderfor cloning and expression studies. To amplify the target gene, conditions were optimized by using the gene specific primers of the interleukin-1Ra gene. Amplified product about 500bp comprises of the coding region of interleukin-1 Ra was cloned in plasmid PCR 2.1. Recombinant was confirmed by analyzing through colony PCR and restriction digestion. Sequence verification of the gallbladder cDNA derived clone showed 100% homology with the reported sequence of interleukin-1Ra. Another part of the current study that presented in the thesis is to deal with some modification to interleukin-1Ra gene by site directed mutagenesis and to study the effects of these modifications on protein expression, solubilization, refolding and their effects on biological activity as compared with the wild type recombinantinterleukin-1receptor antagonist (rhIL-1Ra). As interleukin-1Ra contain four cysteine residues at position 66, 69,116 and122(Schreuder et al. 1995). Two cysteine residues 69 and 116 are involved in disulphide bridge formation and are responsible for biological activity of the interleukin 1Ra while cysteine at 66 and 122 positions are not involved in disulphide bridge formation. So it was proposed that substitution of these free cysteines with serine amino acid may be helpful to generate recombinant interleukin-1Ra mutants with more homogenicity, high reproducibility and with enhanced specific activity. By performing site directed mutagenesis replaced the cysteine 66 to alanine, cysteine 66 to serineand both cysteine 66& 122 replaced to serine and similarly delete the cysteine residue at position 66, both 66&122, and 116. Screen the positive mutated vi clone with sequence verification. In order to evaluate the expression study in bacterial system the above positive mutated clones (WrhIL-1Ra, Mutant1 (Cys66Ala), Mutant 2 (Cys66Ser), Mutant3 (Cys66&122Ser), Mutant 4 (Cys66 deleted), Mutant 5 (Cys66&122 deleted) and Mutant 6 (Cys116 deleted) were cloned into the expression vector PET 30 under T7 promoter. Different E-coli expression host strains such as, RossettaDE3, BL21DE3PLys, BL21DE3 were used to evaluate the high level expression of interleukin 1Ra protein. A high expression level of IL-1Ra was observed in the E-coli expression host strain Rossetta2DE3 as compared to BL21DE3 and BL21DE3PLys. Fermentation conditions were optimized for high expression yield of IL-1Ra. High expression levels and cell biomass were observed in both M 9 modified and auto induction media. Usually the over expressed protein in eukaryotic system is unable to fold in proper conformation and are deposited as insoluble inclusion bodies (IBs). To obtain the active protein from these aggregates conditions were optimized for inclusion bodies isolation, solubilization, refolding and purification. Refold the solubilized inclusion bodies. The refolding yield of rhIL-1Ra mutated clone (as analyzed byRP-HPLC) 60%(Mutant 1, Mutant 2 and Mutant 3), 70% (Mutant 4), 75%(Mutant 5) and 50%(Mutant 6) respectively was observed. Prior to the ion exchange chromatography refolded protein of all the mutants were diafilterd to remove the salts and refolding additives. RhIL-1Ra and its mutant proteins were further purified using AKTA system by ion exchange on DEAE Sepharose column. Purified protein with a yield (WT 300mg/L, Mutant1 (Cys66Ala) 300mg/L, Mutant2 (Cys66Ser) 312mg/L, Mutant3 (Cys66&122Ser) 327mg/L, Mutant 4 (Cys66 deleted) 343mg/L, Mutant 5 (Cys66&122 deleted) 390mg/L and Mutant 6 (Cys116 deleted) 213mg/L respectively was obtained (with 98% purity) as characterized by SDS-PAGE, HPLC and western blot analysis. Biological activity of rhIL-1Ra was calculated by inhibition results of vii thymocyte proliferative response to rhIL-1Ra as described (Tan et al. 2005). Purified protein of all the mutant shows inhibition of thymocyte proliferation to some extent but when compare to wild type IL-1Ra no significant difference in activity was observed in the wild type and in Mutant1 (Cys66Ala). It was observed that biological activity of the Mutant 2 (Cys66ser) and Mutant 3 (Cys66 &122 Ser) displayed 3 fold and 7 fold higher activity than the wild type IL-1Ra. Mutant 4 (Cys66 deleted) and Mutant5 (Cys66 &122 deleted) although express high yield protein, but dramatically had 5 fold and 10 fold low activities as compared to wild type IL-1Ra. While Mutant 6 (Cys116 deleted) showed 50 fold less activity. In Conclusion we constructed six mutants (Cys66Ala, Cys66Ser, Cys66&122Ser, Cys66 deleted, Cys66&122deleted, Cys116deleted) by site-directed mutagenesis and characterized. Optimization in expression and purification process significantly enhanced the expression and product yield of the some mutants of rhIL-1Ra. These modifications enhance the mutants (Cys66Ser, Cys66&122Ser) activity as compared to the native rhIL 1Ra. The strategy applied here may also be helpful to express and purify other functional therapeutic proteins