Two or more similar DNA sequences can be digested

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DNA shuffling is a process by which two or more similar DNA sequences can be digested using restriction enzymes which cuts the DNA fragments in the same place. These fragments are mixed together using ligation reaction and this ultimately gives rise to a large number of hybrids. It is done so that the hybrids produced have unique properties or activities i.e., high activity and thermostability which were not present in the original sequences. In this case, DNA shuffling is used to generate a new hybrid cystatin which contains improved cathepsin B inhibitory activity. It is obtained from canecystatin-1 and oryzacystatin-1, which on recombination gives rise to the hybrid cystatin.

This technique of DNA shuffling differs from error-prone PCR because of the following reasons.

In error-prone PCR, DNA polymerase is used to amplify the target DNA by PCR which results in the insertation of incorrect nucleotides into the replicating DNA whereas in DNA shuffling, similar genes encoding different properties are shuffled together to produce different hybrids.

The method of error-prone PCR has a high error rate when the polymerase is used to amplify the wild-type sequence, but in case of DNA shuffling there is no error, when it treated with restriction enzyme.

Error-prone PCR is very effective when a library of mutants of the target gene is constructed whereas DNA shuffling is effective in creating unique properties or activities in the hybrid proteins, which are not present in the original sequences.

The gene shuffling technique is more suitable because the two genes CaneCPI-1and OC-I are different, but present high similarity in their DNA sequence. The gene CaneCPI-1was isolated from sugarcane, whereas the gene OC-I was extracted from rice and both of them reveal a similarity of 56%. These two genes were used in the library construction. The PCR products used in the reaction was amplified from these two genes using the pET28aCaneCPI-1 and pET28OC-I plasmids, as templates.

(b) What are the properties of DNase I and why was it used (in preference, for example, to a restriction enzyme) to cut the DNA for the gene shuffling procedure? (15 marks)

DNase I is a glycosylated polypeptide enzyme that is used in degradation of single and double-stranded DNA. It helps in the cleavage of DNA into 5´phosphodinucleotide and oligonucleotide fragments. In case of single stranded DNA, the activity of DNase I is much less compared to double stranded DNA. Divalent ions like Mg2+, Ca2+ or Zn2+ modifies the activity of DNase I i.e., DNA is degraded by making single-strand cuts in the phosphate backbone. On the other hand DNase I create double-strand breaks in the presence of Mn2+ or Co2+. They can remove DNA from RNA and protein preparations and can be used to nick translating DNA which can result in the generation of random fragments of DNA. They can cleave the DNA strand of a DNA: RNA hybrid and keep the RNA strand intact. They do not digest DNA to mononucleotides but yield 5'-phosphate di- and tri-nucleotides.

In the experiment conducted, DNase I is used as the restriction enzyme to cut DNA because they can cut all the 4 bases in heterogeneous dsDNA and the cleavage induced does not differ more than 3-fold. Apart from this it was seen that DNase I could completely digest the substrates of DNA which was present in 100µl of buffer which contained 50 mM Tris-HCl along with 1 mM MnCl2. The pH of the buffer was maintained at 7.4. The use of Mn2+ instead of Mg2+ in this step also improves the fidelity of DNA shuffling three times. There are other important features of this enzyme and these factors have lead to the usage of D in not only this experiment but also other experimental procedures. They can identify o protein binding sequences on DNA, help to prevent clumping when handled with cultured cells, and leads to the creation of a fragmented library of DNA sequences in case of in vitro recombination reactions.

(c) Describe and explain the methods used for selecting desirable mutant genes? (20 marks)

The mutant genes selected in this experiment are CaneCPI-1and OC-I Site-directed mutagenesis

(d) Explain the evidence that shows why the cystatin mutants described in this paper represent progress towards development of inhibitors of human cathepsin B? (25 marks)

The cystatin mutant genes showed that they are responsible for the inhibition of human cathepsin B when were measured against human cathepsin B and L. It was seen that these cystatin mutants like Oryzacystatin-1, Canecystatin-1, Canecystatin-4b etc showed inhibition in the levels of enzyme in case of cathepsin B and L. The inhibitory activity was measured using the fluorogenic assay Z-Phe-Arg-MCA as substrate. KM values of 2 μM and 23.4μM were presented in case of cathepsin L and B. The hydrolytic activity of the enzyme was measured at different concentration with different inhibitors for 5 minutes. The Ki values of the cathepsin B shows evidence that inhibition of the enzymatic activity had taken place.

Cystatin Ki value of Cathepsin B(nM)

Oryzacystatin-1 78.5

Canecystatin-1 87.6

Canecystatin-4b 0.58

Clone A10 11.2

When the enzyme activity was measured, it was seen that the two clones OC-I NΔ and A10 showed unique characteristics in case of enzyme inhibition. The OC-I NΔ clone was derived from oryzacystatin-1 and it showed no activity in case of cathepsin B but in case of cathepsin L it showed medium activity. The A10 clone on the other hand showed inhibitory activity in case of both the enzymes, though its activity was mainly high in case of cathepsin B. Another fact was revealed which showed the binding capacity of cathepsin L to be more than cathepsin B as the cystatin Oryzacystatin-1 and Canecystatin-1 bind more tightly to it. There are several differences that can be noticed between A10 and canecystatin-1. The first difference being that the N-terminal region of A10 is derived from oryzacystatin-1 due to gene shuffling process. Secondly, a 7 amino acid deletion is seen in the N-terminal region. Lastly, two unexpected point mutations I30T at the beginning of the α-helix and L97Q in strand β5 are seen. The A10 clone shows a lot of peculiarities in enzyme inhibition. The two reverse mutants of A10 (T30I and Q97L), cannot inhibit Cathepsin B. However, both of these mutants can easily inhibit Cathepsin L at the nMolar level. But the more surprising feature is the ability of A10 to inhibit cathepsin B at all. The accumulation of the two mutation somehow overcome the deleterious effect of the N-terminal deletion and turn A10 into a nMolar inhibitor of cathepsin B.

(e) What obstacles remain to be overcome before mutants of cystatin can be introduced into clinical medicine? (20 marks)

On studying the model of canecystatin-1 and A10, several problems were revealed in their inhibitory mechanisms. The canecystatin-1 structure comprises of single α-helix and five-stranded anti-parallel β-sheet and a hydrophobic core. There are three active sites in phytocystatins which interact with binding sites in the enzyme. The first interaction occurs in the canecystatin-1 residues 59-63. The second interaction is between V90 and W91 (P 83 and W84 in oryzacystatin-1) and the third interaction site is formed by the N-terminal region. These interacting sites help in creating an interaction surface between cystatins and cysteine proteinases. But due to differences between cathepsin B and cathepsin L, the entry of all the three elements into the inhibitor's active site is prevented. Recent studies with single mutations confirm that the N-terminal region of phytocystatins has a lot of importance. It is shown that the N-terminal deletion mutant and the double reverse mutant, are unable to inhibit cathepsin B. But the two point mutations which activitate A10 activate indirectly because they are located far away. The first mutation (I30T) changes the hydrophobic cluster formed by residues F50, L53, I30 and the second mutation (L97Q) disrupts the opposite side of the hydrophobic core formed by L32, F35, A36,V86, F100 and L97. These mutations get released when they destabilize the hydrophobic region which ultimately affects the N-terminal region and the remaining two loops. The N-terminal region is quite flexible which allows A10 mutant to bind to the enzyme. The cystatins assemble itself into different oligomeric states and form amyloid fibrils due to the swap of three-dimensional domain within the cystatin family. This hypothesis gets support from the fact that A10 is less soluble than the parent molecules and aggregates as inclusion bodies when heterologously expressed. When these mutants were studied based on their inhibitory action, it shows that both mutations are necessary for the increased activity of A10 towards cathepsin B, though A10 is less active towards cathepsin B compared to other cystatins. Thus, the above hypothesis can be used for the development of tighter binding cathepsin B inhibitors. Sugarcane can be used to develop many inhibitors other than canecystatin-1. Canecystatin-4 which is also derived from sugarcane has a similar binding capacity as that of cystatin-C. It shows a wide range of variations in the hydrophobic residues and aggregates more quickly than canecystatin-1. Thus, these obstacles need to be overcome and tight binding cathepsin B inhibitors should be developed before mutants of cystatin can be introduced into clinical medicine.