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How does gene shuffling differ from error-prone PCR. DNA shuffling which is known also as "sexual PCR" is one of most advanced technologies exploited for generation of randomized libraries of mutant genes by using DNase I enzyme to fragment the DNA ,on the other hand error prone PCR is one of the commonly used approaches to generate libraries of random mutants of nucleotide sequences and also for introduction of point mutations in sequential way by utilizing the ability of Taq polymerase, to introduce random errors during the prolongation of the new DNA strand allowing deleterious mutations to be accumulated in conjugation with beneficial ones, the other important difference is rate of introducing point mutation ; it has been noticed that error prone-PCR is associated with relatively high rate of point mutations (0.7%) in comparison with gene shuffling which is characterized by very low rate of point mutagenesis (0.05%) especially with addition of divalent cation (Mg+2,Mn+2) ( Pritchard, et. al, 2005) .
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Why was the gene shuffling technique more suited to the application desribed in the paper?
Gene shuffling technique is favored in this paper to select the distinct gene with the similar DNA sequence such as canecystatin-1 and oryzacystatin-1, to construct a new hybrid mutant of cystatins with enhanced inhibitory activity toward cathepsin B., which makes DNA shuffling more suitable for introduction of mutations to produce new hybrid of cystatins (i.e enhanced possibility for evolutionary gene) is due to the characteristic feature of block changes typical of gene shuffling which enable the removal of deleterious mutation by back-crossing with parental DNA while error prone PCR is only able to produce point mutations but do not have the feature of eliminating deleterious mutations which is necessary in the case of developing cystatins hybrids (Cooper and Cass 2004) in addition gene shuffling is a directed evolution process that rely on in vitro selection and formation of huge library of chimeric mutated genes from a set of parental ones so that the desired function of the selected recombined proteins remains the same (i.e becomes the parent for the next generation) (Zhao and Arnold, 1997).
What are the properties of DNaseI
(b) Deoxyribonuclease I (DNase I) is a 30,400 Da endonuclease glycoprotein, which is used as footprinting agent to map proteins on genomes, and investigating variations in the DNA structures , this enzyme has numerous factors that enable it to interact very well with DNA molecules and these include positively charged amino acid side chains on the exposed loop of the enzyme that interact with the phosphates on both sides of the DNA minor groove, van der Waals interactions also occur in the DNA minor groove, assisted by tyrosine 76 and arginine 41in the protein region, which are both crucial for DNase I activity , DNase I enzyme responsible for cleavage of the phosphodiester bond in the DNA double helix controlled by presence of divalent cations (Ca +2, Mn+2 ) , leading to introduction of single-stranded nicks by cleaving the P-O3â€² bond.(Fish and Vournakis 1987 , Heddi et al. 2009)
why was it used (in preference to a restriction enzyme) to cut the DNA for the gene shuffling procedure?
DNAse I endonuclease is a type of restriction enzymes that include as well type II and type III endonuclease but DNAse I is the used to control size of the DNA fragment by cutting it into pieces of 50-100 base pair (Cohen 2001), DNAse I is preferred as it cut the homologus fragments of DNA into similar pieces which will anneal to each other so that hybrids can be produced and used to generate a library of mutants with characteristic properties.
Another reason for the preference of DNAse I is its structural specificity to the DNA molecules by interacting with minor grooves depending on the width of these grooves (Fish and Vournakis 1987).
(c) Describe and explain the methods used for selecting desirable mutant genes
Different techniques are used nowadays to generate genes with desirable and unique properties via mutation and these techniques can be coupled to each other or used individually and It involves cloning and analysis, site-directed mutagenesis, recombination and the design of molecular model, which depend on directed evolution that mimics the process of natural development.
cloning and analysis
The plasmid pET28a was dissected by Eco RI and Nde I and dephosphorylated with shrimp alkaline phosphatase (SAP) , the amplified final products were digested and ligated with the synthetic plasmid to switch E.coli ROSETTA (DE3) for expression of the hybrid proteins
dideoxy method was employed to sequence 2000 clones exploiting an ABI Prism 377 (Applied Biosystems) after that several clones were selected after analysis by BLAST database which contains some sequence information for the nucleic acid of DNA sequence and peptide sequence of proteins and with help of Multalin software, and these selected mutants were subjected to expression analysis and subsequent inhibitory activity assays. (Valadares et al. 2010)
Application of site directed mutagenesis enables introduction of mutations almost everywhere within a gene at high levels of efficiency through the use of linear PCR and plasmid techniques ( Reikofski and Tao 1992).
Invitrogen system was applied to perform site directed mutagenesis ,by using the encoding plasmid PET28a for A10 mutant as template DNA for the construction of the reverse mutant 1 (T30I) and mutant 2 (Q97L) after that mutant 3 is developed from mutant 2 as template and mutant 1 as primer, than plasmids were sequenced and analysis of PCR products performed to confirm the correct molecular weight of products, in addition to these three mutants two clones were selected from the shuffling library one of these is obtained form pure oryzacystatin by N-terminal
Deletion of seven residue. The second one obtained by hybridization (clone A10) which contain 2 mutations and N-terminal deletion.
Valadares et al. has exploited Swiss-Prot database for retrieval of amino acid sequences of oryzacystatin-1 and human stefin B and to get the three dimensional structure from protein databank, Subsequently, CLUSTALX can align the sequences of those Therefore, cystatin mutants can be sequenced in term of this template.
The software of MODELLER 9v8 have used by the researchers to compare molecular models corresponding to each of these alignments; statistical methods such as MODELLER and Discrete Optimized Protein Energy (DOPE) score have been applied to evaluate quality of molecular model and predict protein structure according to homology models (wikipedia 2010), further evaluation of The models were also done by the programs VERIFY 3D and WHATIF , followed by selection of a representative model for structural analysis .
(d) Explain the evidence that shows why the cystatin mutants described in this paper represent progress towards development of inhibitors of human cathepsin B
Valadares et al. have developed 7 cystatin mutants (OC-I, CaneCPI-1, CaneCPI-4, OC-I NÎ”, A10, mutant 1, mutant 2 and mutant 3 ) and assessed the inhibitory activity of these mutants by fluorometric assay to measure the hydrolytic activity of the enzyme by using different concentrations of the inhibitors; from these mutants only two clones (OC-I NÎ” and A10) revealed interesting inhibitory activity for cathepsin B and L.
Investigators have focused on A10 mutant which presented increased inhibitory activity towards Cathepsin B which the main interest for the researchers and they have concluded that the reason for this improvement comes from acquisition of two unexpected point mutations in the A10 clone which affect the hydrophobic residues of the protein core I30T at the beginning of the a-helix and L97Q in strand b5,secondly oryzacystatin-1 contribute to the N-terminal region of A10 not canecystatin-1 , last reason is the deletion of 7 amino acids from this region.
The evidence behind improved inhibitory activity of A10 comes from replacement of leucine by glutamine residue at position 97, also the interaction between Î²1 and Î²2 strand near the N-terminus of the helix can construct the hydrogen-bonds as well as the hydrophobic interaction in the A21 and V56 positions, finally I30 and the residues of the conserved LARFAV sequence which are embodied in the helix appears to involved in the hydrophobic core; and the role of this motif according to authors is to provide further stabilization of the tertiary structure by adding complementarity to the hydrophobic residues in the Î² -sheet of the phytocystatins,
Homology model of clone A10 showing the residues correspondent to the point mutations
The three active sites of phytocystatins have been associated with the formation of complex between stefin B and papain for the interaction with the binding pocket of enzymes. The first active point is the 59-63 residues of canecystatin-1( QVVAG) have same conserved sequence of oryzacystatin-1 and mutant A10. The second action site contains V90 and W91 in the canecystatin-1, and P83 and W84 in oryzacystatin-1; The third interaction region is the N-terminus .
Despite of these three elements of the inhibitor's active site; cystatins are impeded from simultaneous entry to cathepsin B due to the presence of occluding loop that covers part of the binding pocket; on the other hand activity of A10 clone is restored by the two point mutations which are located distant from the active site loops.
These first point mutations I30T are expected to be correlated with the destabilization of the hydrophobic clusters and loop binding the N-terminus of the helix while second mutation (L97Q) is believed to dislocate the opposite side of the hydrophobic core, according to these point mutations destabilization of the hydrophobic core will lead to its release either partially or completely. This release will cause decoupling of N-terminus and the two loops on the other active site of the inhibitor leading to enhanced flexibility of the N-terminal region of the A10 mutant to reserve its ability to bind to enzyme, Although of the low activity of A10 towards cathepsin B compared with other natural cystatins such as cystatin C, but still there is a rational basis which for its usage in the development of inhibitors with tighter binding ability to cathepsin B.
(e) What obstacles remain to be overcome before mutants of cystatin can be
introduced into clinical medicine?
Mainly two essential problems are facing researcher that will try to introduce cystatins into clinical medicines which are they:
1- the unstable interaction at the interface which is caused by disturbance of the hydrophobic contacts between the secondary structural elements of cystatins with the hydrophobic core of cathepsin B.
2- Solubility of the developed mutants like A10 clone is much more less than the original cystatins so that exposure of its hydrophobic core will lead to its aggregation into inclusion bodies.
the importance of developing mutants of cystatins with improved inhibitory activity towards cathepsin B arises from the evidences that reveal strong involvement of cysteine protease as causative risk factor for cancers and neurodegenerative diseases so this might indicate that cystatin mutants can have potential anticancer and psychoactive agents, However, only a few of small-molecular mutants of cystatin proteases have been investigated in the preclinical trials for their possible role as biological therapeutics.
the complexity of the three dimensional structre of cathepsin B in the tumor cells and tissues make difficult to target the inhibitors to the active site, some inhibitors show effective in vitro potency, but it show significantly reduce therapeutic effect in vivo trials, This inconsistency resulted from the difference of the culture system in vitro and real tumor microenvironment, so it minimize the chance to reproduce the same effect of inhibitor in the normal organism.( Palermo and Joyce 2007)