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The Bioinformatic field is one of the most developing fields in today’s biotechnology’s world. A number of different online DNA and protein alignment tools and databases provided by biotechnical institutions such as Uniprot (Universal Protein Resource), NCBI (National Center for Biotechnology Information) and EBI (European Bioinformatic Institute) expands the opportunities for a more reliable and less labouring research. As for this study, in order to study the homology of the MO2.1 protein and studying its sequence identity with other proteins available in online protein databases bioinformatic tools such as Fasta, Blast, and Jalview was used.
The tertiary structure of a protein is a crucial element in determination and localization of the function of the protein . However at the time this work was carried out no actual tertiary structure was yet available for MO2.1. Therefore in order to study the homology of MO2.1 with other AMPs the MO2.1 protein sequence (QGPGRQPDFQRCGQQLRNISPPQRCPSLRQAVQLTHQQQGQVGPQQVRQMYRV ASNIPST) was first aligned against PDBs (Protein Data Banks) available in Uniprot protein database using BLAST (Basic Local Alignment Search Tool). Based on the results from blast, the MO2.1 protein (Uniprot ID: 24303), was multiple aligned with Mabinlin-1, Napin, and 2s albumin.
In order to direct the mutagenesis PCR, a total of 5 mutagenic primers were designed manually in accord with the instructions in the Instruction Manual for mutagenesis PCR7. The primers were synthesised at Eurofins MWG Operons.
Using multi site-direction mutagenesis made it possible to employ up to 3 primers in each reaction while each of the primers could contain up to 5 different mismatches. The Table 1 shows the map for all positions and numbers in both DNA and protein sequence where mismatches have been directed for both MUT1-MO2.1 and MUT2-MO2.1.
In order to make fresh culture of the E. coli cells containing TOPO-MO2.1, a volume of 200 « l of the cell culture was added to a total volume of 5 ml LB medium (Lysogeny Broth) ( See ! » »# »$%&'(‘ »‘)*’%+#, »*’%)#-%(#,)./ Error! Reference source not found.) in a 15 mL falcon tube. The antibiotic Kanamycin (100 « g/ »l) was added to a final volume of 50 (« g/ml). Since the TOPO plasmids contain the Kanamycin resistance gene the antibiotic Kanamycin is used to provide a safe growth for these TOPO containing E. coli cells by inhibiting any other bacterial growth. The medium was then incubated at 37 °C for <16 hours.
For this step the QIAprep Spin Miniprep Kit from QIAGEN Figure 4, was applied.10 The fresh culture from previous step was transferred in 2#1.5 ml eppendorf tubes and centrifuged at 7000 rpm for 5 minutes in a table microcentrifuge. The supernatant was discarded and the remaining pellet bacterial cells were suspended in 250 « l Buffer P1 (provided with the kit). 250 « l of the Buffer P2 was added and mixed thoroughly by inverting the tubes 4-6 times. Next 350 « l of Buffer N3 was added and mixed immediately and thoroughly by inverting the tubes 4-6 times. The tubes were then centrifuged at 13000 rpm for 10 minutes in a table microcentrifuge. The supernatant from this step was applied to the QIAprep spin column (provided with the kit) by pipetting and centrifuged for 60 seconds. The flow-through was discarded and the QIAprep spin column was washed with 0.5 ml of Buffer PB and centrifuged for 60 s. The flow-through from this step was discarded. The QIAprep spin column was then washed again this time with 0.75 ml Buffer PE and centrifuged for 60 s. The flow-though was discarded and the tubes were centrifuged for another 1 minutes to remove remaining wash buffer. For the DNA elution step the QIAprep spin columns were first placed in clean 1.5 ml eppendorf tubes. 50 « l Buffer EB was added to the center of each QIAprep spin column incubated for 1 minute at RT and centrifuged for 1 minute. The Flow-through containing the MO2.1 gene stored at -20 °C for future use .
Using site-directed mutagenesis (SDM) makes it possible to apply changes in form of insertion, deletion at desired sites in the DNA sequence. In addition multi site-directed mutagenesis expands the possibility to make more than one mismatch in one round of reaction .
In the first recombinant MO2.1 the general primary structure (XXYY)n which has previously been applied for synthetic antimicrobial peptides was chosen for alteration of the first !-helix in the MO2.1 peptide. The X in the formula represents the hydrophobic residues, Y represents the positive charged residues, and the n is the number of repeats which can be between 2 to 4. Antimicrobial peptides containing !-helix with this set of repeats have shown inhibitory effects against both gram positive bacteria and yeasts . This pattern has shown to have the best activity when the number of repeats are n = 3  however in this study in order to avoid conformational changes as much as possible the number of repeats was decided to be n = 2.
As it was reported by (Suarez et al.) the MO2.1 protein have shown an increased antimicrobial activity in the part of the sequence where two proline residues are next to each other in the loop between !-helix 1 and 2. With this presuppose that the addition of more hydrophobic residues may improve the bactericidal activity of the protein, more hydrophobic proline (P) and leucine (L) residues were added to the part of the sequence that showed best bactericidal activity according to the results obtained in previous studies by other groups .
In the second set of mutations, the amino acid serine (S), right before the two proline residues which have previously been reported to have an impact on the antimicrobial activity of MO2.1 was replaced with a glutamine residue which improves the coagulation activity. This was done to investigate whether it would change the antimicrobial activity for better or worse. Next, the first two residues in the second !-helix (H2), was replaced with positively charged arginine (R). Although the proline residue in the beginning of the helix does not affect the conformation of the helix  but since it is known to be a poor helix former it was preferably replaced with an arginine. The amino acid serine (s) is also among those residues which are poor in !-helix formation  hence was replaced with arginine. The latter amino acids were both replaced with arginine residue with the expectation of inducing a higher coagulation activity into the protein by having to positive charges next to each other.
Site-directed mutagenesis PCR was used to apply mutations into the DNA sequence of MO2.1. The QuickChange Multi Site-Directed Mutagenesis Kit was supplied from Agilent Technologies and the structures were followed as explained in Instruction Manual11. As it has been reported by (Lei Zheng et al.), QuikChange site-directed mutagenesis is a very efficient method for PCR amplification. This method reduces the chances for dimerization and self-annealing of primers with the restriction that the primers should be within the size distribution of 25-45 bases in length and the melting temperature (Tm) $ 75 °C .
Blue/white screening is a common method used for screening and confirmation of a successful cloning. The principle of this method is based on the fact that in TOPO plasmids the cloning site is located on the lacZ gene. This gene can be used as a reporter gene in a sense that if the target DNA (in this case recombinant MO2.1) is successfully inserted into this site where lacZ gene is located the expression of this gene is then disrupted. As a result when the host cells containing clone-plasmid are spread on the agar plate containing the inducer isopropyl-%-D-thiogalactoside (IPTG) and the artificial substrate 5-bromo-4-chloro-3-indoxyl-%-D-galactopyranoside (X-gal) (used as a colorimetric substrate) there will only be white colonies appearing on the agar plate. However if the cloning has failed the lacZ gene will be expressed hence the %-galactosidase is produced which in presence of the IPTG will metabolize the X-gal to produce a blue colour. In latter case the bacteria colonies containing only the plasmid will appear in blue colour .
Subculture of Colonies
For further studies of MUT1-MO2.1 and MUT2-MO2.1 a total of 100 and 60 white colonies (50%) from each of the two mutant plates respectively were subcultured on Kanamycin LB agar plate (Figure 13). The colonies were chosen from the plate which had a volume of 10 « l of cloned cells loaded on. The plates were incubated at 37 °C for <20 hours and stored at 4 °C for future use.
A volume of 5 ml fresh overnight cultures made from each subcultured colony (see Inoculation Procedure). Each culture was then transferred into 2#1.5 ml eppendorf tubes and centrifuged at 7000 rpm for 5 minutes in a table microcentrifuge. The supernatant was discarded and the pellet was resuspended in 1ml lysis buffer 1#TE buffer (pH=8.4) (! » »# »$% &'(‘ »‘)*’% +#, »*’% )#-% (#,)./, Error! Reference source not found.), and 5 « l lysozyme (50 « g/ »l). The cells were lysed by gently pipetting in and out, incubated in a heat bath at 100 °C for 3 minutes and kept at RT for 15 minutes. The samples were then centrifuged at 13000 rpm for 10 minutes and the supernatant containing crude extract of recombinant MO2.1 was collected and stored at 20 °C for future use.
Protein Concentration Using Bradford Assay
After isolation of the protein the conventional Bradford assay was used for quantification of protein content in each sample. The Bradford assay is a simple and accurate method for estimation of protein concentration in a sample with unknown protein concentration. The method is based on the binding of the dye Coomassie Brilliant Blue G-250 to the protein in the sample. The coomassie dye in cationic form in red and green colour (Figure 6) has an absorbance maximum at 470 – 650 nm respectively. The blue coloured anionic form of the dye (Figure 6) has a maximum absorbance at 595 nm which is when it bonds to the protein. Therefore an absorbance measurement at 595 nm reveals the quantity of the protein-bonded Coomassie Blue which is proportional to the amount of the protein in the sample. The Bradford assay used in this study was acquired from Bio-Rad13. Bovine serum albumin (BSA) (1mg/ml) was used in the concentrations of 2, 4, 6, 8, and 10 (« l) as the standard (! » »# »$%&'(‘ »‘)*’%+#, »*’%)#-%(#,)./ Error! Reference source not found.). For each sample a volume of 5 « l of the protein sample Error! Reference source not found.) was transferred into a 1 ml polystyrene cuvette (10#4#45mm), 200 « l of Bradford reagent was added to each cuvette and the volume was adjusted to 1 ml by distilled water. The content of the cuvette was then mixed gently in order to homogenize the reaction mixture and kept at room temperature for 5 minutes. The absorbance was measured at 595 nm using UV-Visible spectrophotometer. The unknown concentration of each sample was then calculated using the formula: Abs = [C] # M.
Table of contents :
2.! Materials and Methods
2.2.! Inoculation Procedure
2.3.! Plasmid Isolation
2.4.! Site-Directed Mutagenesis
2.4.3.! Mutagenesis PCR
2.5.! Blue/White Screening
2.6.! Subculture of Colonies
2.7.! Protein Isolation
2.8.! Protein Concentration Using Bradford Assay
2.9.! Protein Activity
2.9.1.! Coagulation Activity Using Clay Solution
2.9.2.! Antimicrobial Activity
2.9.3.! Disc Diffusion Method
3.! Results and Discussion
3.2.! Mutagenesis PCR
3.3.! Coagulation Activity
3.4.! Antimicrobial activity
3.4.1.! Disk Diffusion Method
3.6.! Protein Concentration