Title of Invention	PROCESS OF PRODUCING A NOVAL ANTIMICROBIAL PEPTIDE"
Abstract	A synthetic oligonucleotide was constructed, cloned in pBADgIIIA and expressed in E. coli TOP 10. This unprocessed peptide was purified using Ni-NTA column. The purified unprocessed peptide was cleaved to generate processed bioactive peptide . This peptide which is 16 amino acids in length rich in Lysine and Alanine amino acids is a hydrophobic alpha helical peptide which exhibited anti microbial activity without any haemolytic activity.

Title of Invention

PROCESS OF PRODUCING A NOVAL ANTIMICROBIAL PEPTIDE"

Abstract

A synthetic oligonucleotide was constructed, cloned in pBADgIIIA and expressed in E. coli TOP 10. This unprocessed peptide was purified using Ni-NTA column. The purified unprocessed peptide was cleaved to generate processed bioactive peptide . This peptide which is 16 amino acids in length rich in Lysine and Alanine amino acids is a hydrophobic alpha helical peptide which exhibited anti microbial activity without any haemolytic activity.

Full Text	2. Prior Art: Antimicrobial peptides are produced by both invertebrates and vertebrates. These peptides seem to play a critical role in the defense mechanism of these animals. Dermaseptins are a class of antimicrobial peptides, whose size range from 28-40 amino acids. They are produced by tree frog Phyllomedusa bicolor. Krugliak et al, [2000] have derived synthetic constructs from dermaseptins that are smaller than native peptide which still exhibited antimicrobial activity. Earlier patents for short bioactive anti microbial peptides were claimed by Owen, Donald R, 1^^ May, 2003 [US 20030083243] Here we describe designing an alpha helical alanine and lysine rich peptide which is different from lysine rich native peptide reported earlier. The designed peptide has higher hydrophobicity when compared to that of the native. The designed peptide exhibits antimicrobial activity and is not hemolytic [Figure 1]. 3. Description of the invention : Consist of the following steps: Annealing of the synthetic oligonucleotide strands: 5' TC GAG CGC GCG CTG TGG AAA AAC ATG CTG AAA GCG CTG GCG AAA ATT GTG GCG CGC CCA 3'. 1'^ strand = 59 nucleotides 3' C GCG CGC GAC ACC TTT TTG TAC GAC TTT CGC GAC CGC TTT TAA CAC CGC GCG GGT TCG A 5'. 2"^ strand = 59 nucleotides 1) Equal aliquots of both the strands are taken. 2) The mixture was kept at 95° C for 4 minutes in dry bath. 3) It was gradually allowed to cool to room temperature. 4) The strands used for annealing were 100 \|ig /ml concentration. Double digestion of p BAD A vector with Xhol and Hindlll Restriction enzymes. Reaction mix 1. 1 OX Buffer : 12^1 [composition: 50mM NaCl, lOmM Tris.HCl, lOmM MgCb, ImM DTT pH 7.9] 2. Plasmid DNA : 60 ix3. Enzymes Xhol [20,000U/^1] : 3^1 Hindm [20,000U/^1 : 3\|il Total volume : lOOp.1 Incubation time (37° C) is 4hrs. Purification of the digested vector was done usu chloroforai-isoamyl alcohol [24:1] The digestion of the vector was confiraied using 0.7 % agarose gel electrophoresis wi uncut vector DNA as control. Ligation of the double digested vector with annealed fragments Reaction mix: 1. Annealed fi-agments : 5 \x 2. T4DNA ligase buffer [lOX] : 5 \|il [50mM NaCl, lOOmM Tris.HCl, lOmM MgCb, 0.025% Triton-X-100] 3. Double digested purified pBAD-A : 5 \|il 4. T4 DNA ligase [400,000U/^1] : 1 ^il 5.MQ : 34 ^l The Reaction mixture was incubated for 1 hr at 37°C. Transformation of ligated vector in E.coli ToplO. Competent cell preparation 1) 1ml of mid log Exoli TOP 10 was taken in a microcentrifuge tube. 2) Centriguged at 4000 rpm for 5 minutes at 4° C. 3) The pellet was resuspended in 750\|il of 10 mM calcium chloride a incubated in ice for 40 minutes. 4) After incubation it was centrifuged at 2000xg for 5 minutes at 4° C. 5) The pellet was re-suspended in 150^1 lOmM ice cold calcium chloride. 6) The suspension was stored in ice for a maximum of 4 hours. Transformation 1) 2 ^l of ligated mix was added to the competent cells and mixed well. 2) The tube was incubated in ice for 30 minutes. 3) The tube was then kept in 42°C water bath for 120 seconds. 4) To this 150\|j,l of LB broth was added and incubated at 37° C for 30 minutes. 5) From this 100 ^1 aliquots were spread on LB agar medium containing 25^g/ml ampicillin and incubated at 37^0. 6) Expression of cloned DNA was done using Tris /Tricine polyacrylamide gel. 7) The peptide was over expressed using arabinose induction. Arabinose induction of clones - - 1) 1ml of over night saturated culture was added to 15 ml of LB with Ampicillin 25\|ig/ml and Streptomycin 20^g/ml. 2) This was incubated at 37°C till it reached mid log phase. 3) From this .1.5ml of culture was withdrawn and kept in ice and this served as un-induced culture. 4) 15^1 of 20% arabinose (Hi-Media) was added to the rest of the culture and incubated at 37°C for 10 hours. 5) After induction time, the cells were pelleted down in a microcentrifuge tube. 6) Suspended the pellet in 100)il of sample solubiiizing Buffer. The tubes were kept in boiling water bath for 5 minutes and the samples were loaded on a 20% Tris /Tricine Polyacrylmide gel. Purification of 6X his tagged unprocessed peptides by Ni-NTA Qiagen Column 1) The arabinose induced cells were pelletted and resuspended in 1ml of buffer B. 2) The cells were incubated with agaitation for 1 hour at room temperature. 3) The cell lysate was centrifuged at lO^OOOxg for 20-30 minutes at room temperature to pellet down cellular debris. The supernatant was collected. 4) The Ni-NTA column was pre equilibrated with 600)11 of Buffer B and centrifuged for 2 minutes at 7000xg. 5) Up to 600\|-il of cleared lysate supernatant containing the 6X His tagged unprocessed peptide was loaded onto the pre-equilibrated Ni-NTA spin column. It was centrifuged for 2 minutes at 7000xg and the flow through was collected. 6) Ni-NTA column was washed with 600 \|il of buffer C, centrifuged for 2 minutes at 7000xg and the eluate was collected. 7) The eluates were separated on 20 % Tris/Tricine Polyacrylamide gel. 8) To 100\|il of unprocessed peptide purified by Ni-NTA column, lOjal of buffer (IM Tris pH 7.7) was added. 9) 1 \|j.l [50ng] of the Endoproteinase Arg-C was added. 10) Incubated for 5hrs at 37 °C. Spectrophotometric Assay for Antimicrobial Activities [figure 1] 1) 10^ cells were inoculated in 2X - TY medium Tryptone : 16g/li Yeast extract : lOg/li Sodium chloride : 5g/li 2) The cells were allowed to grow till mid-log phase. 3) lOOfil of bacterial suspension was mixed with 100\|il peptide suspension and mixed. Another aliquote of 100\|il bacterial suspension was mixed with lOOjil of plain culture medium and this served as control. 4) Inhibition of proliferation was determined by OD measurements (620nm) after incubation period of 1 to 4 hours. HEMOLYTIC ASSAY 1) Erythrocytes were isolated from heparinized blood by centrifugation . 2) Cells were washed thrice with Phosphate buffer saline (150mM KCl, 5mM Tris HClpH7.4,0.15MNaCl). 3) Erythrocytes were suspended in 10 volumes of PB S buffer (stock cell suspension). 4) The stock cell suspension was diluted 25 fold with PBS buffer and incubated in the water bath at 37°C for 15 min. 5) The test sample was added with antimicrobial peptide and incubated for Ih. 6) The samples were centrifuged at 4000xg for 5 min and the absorbance of supernatant was determined at 540nin. We claim: 1. a process of bioproduction of antimicrobial peptide which has the amino acid sequence ALWKNMLKALAKIVAR (single letter codes of amino acids) exhibiting antimicrobial activity without hemolytic activity, the said process comprising the steps of: a) making a construct of nucleotide sequence encoding 17 aminoacid peptide of the sequence " Argine - Alanine - Leucine - Trypto phan - Lysine - Asparagine - Methionine - Leucine - Lysine - Alanine - Leucine - Alanine - Lysine - Iso leucine - Valine - Alanine - Arginine", b) cloning the coding fragment in frame in pBAD g IIIA plasmid, c) expression of the cloned fragment by cultivating the transformed E, coli in arabinose-containing medium, d) purification of the said peptide using Ni-NTA affinity column and e) cleaving the purified peptide using clostripain to give a peptide sequence of Alanine - Leucine - Tryptophan - Lysine - Asparagine - Methionine -Leucine - Lysine - Alanine - Leucine - Alanine - Lysine - Iso leucine -Valine - Alanine - Arginine.

Full Text

2. Prior Art:
Antimicrobial peptides are produced by both invertebrates and vertebrates. These peptides seem to play a critical role in the defense mechanism of these animals. Dermaseptins are a class of antimicrobial peptides, whose size range from 28-40 amino acids. They are produced by tree frog Phyllomedusa bicolor.
Krugliak et al, [2000] have derived synthetic constructs from dermaseptins that are smaller than native peptide which still exhibited antimicrobial activity. Earlier patents for short bioactive anti microbial peptides were claimed by Owen, Donald R, 1^^ May, 2003 [US 20030083243]
Here we describe designing an alpha helical alanine and lysine rich peptide which is different from lysine rich native peptide reported earlier. The designed peptide has higher hydrophobicity when compared to that of the native. The designed peptide exhibits antimicrobial activity and is not hemolytic [Figure 1].
3. Description of the invention :
Consist of the following steps:
Annealing of the synthetic oligonucleotide strands:
5' TC GAG CGC GCG CTG TGG AAA AAC ATG CTG AAA GCG CTG GCG AAA ATT GTG GCG CGC CCA 3'. 1'^ strand = 59 nucleotides
3' C GCG CGC GAC ACC TTT TTG TAC GAC TTT CGC GAC CGC TTT TAA CAC
CGC GCG GGT TCG A 5'. 2"^ strand = 59 nucleotides
1) Equal aliquots of both the strands are taken.
2) The mixture was kept at 95° C for 4 minutes in dry bath.
3) It was gradually allowed to cool to room temperature.
4) The strands used for annealing were 100 |ig /ml concentration.

Double digestion of p BAD A vector with Xhol and Hindlll Restriction enzymes. Reaction mix
1. 1 OX Buffer : 12^1
[composition: 50mM NaCl, lOmM Tris.HCl, lOmM MgCb, ImM DTT pH 7.9]
2. Plasmid DNA : 60 ix3. Enzymes Xhol [20,000U/^1] : 3^1
Hindm [20,000U/^1 : 3|il
Total volume : lOOp.1
Incubation time (37° C) is 4hrs. Purification of the digested vector was done usu chloroforai-isoamyl alcohol [24:1]
The digestion of the vector was confiraied using 0.7 % agarose gel electrophoresis wi uncut vector DNA as control.
Ligation of the double digested vector with annealed fragments
Reaction mix:
1. Annealed fi-agments : 5 \x
2. T4DNA ligase buffer [lOX] : 5 |il
[50mM NaCl, lOOmM Tris.HCl, lOmM MgCb, 0.025% Triton-X-100]
3. Double digested purified pBAD-A : 5 |il
4. T4 DNA ligase [400,000U/^1] : 1 ^il 5.MQ : 34 ^l The Reaction mixture was incubated for 1 hr at 37°C.
Transformation of ligated vector in E.coli ToplO.
Competent cell preparation
1) 1ml of mid log Exoli TOP 10 was taken in a microcentrifuge tube.
2) Centriguged at 4000 rpm for 5 minutes at 4° C.
3) The pellet was resuspended in 750|il of 10 mM calcium chloride a incubated in ice for 40 minutes.
4) After incubation it was centrifuged at 2000xg for 5 minutes at 4° C.
5) The pellet was re-suspended in 150^1 lOmM ice cold calcium chloride.
6) The suspension was stored in ice for a maximum of 4 hours.

Transformation
1) 2 ^l of ligated mix was added to the competent cells and mixed well.
2) The tube was incubated in ice for 30 minutes.
3) The tube was then kept in 42°C water bath for 120 seconds.
4) To this 150|j,l of LB broth was added and incubated at 37° C for 30 minutes.
5) From this 100 ^1 aliquots were spread on LB agar medium containing 25^g/ml ampicillin and incubated at 37^*0.
6) Expression of cloned DNA was done using Tris /Tricine polyacrylamide gel.
7) The peptide was over expressed using arabinose induction.
Arabinose induction of clones - *-
1) 1ml of over night saturated culture was added to 15 ml of LB with Ampicillin 25|ig/ml and Streptomycin 20^g/ml.
2) This was incubated at 37°C till it reached mid log phase.
3) From this .1.5ml of culture was withdrawn and kept in ice and this served as un-induced culture.
4) 15^1 of 20% arabinose (Hi-Media) was added to the rest of the culture and incubated at 37°C for 10 hours.
5) After induction time, the cells were pelleted down in a microcentrifuge tube.
6) Suspended the pellet in 100)il of sample solubiiizing Buffer. The tubes were kept in boiling water bath for 5 minutes and the samples were loaded on a 20% Tris /Tricine Polyacrylmide gel.
Purification of 6X his tagged unprocessed peptides by Ni-NTA Qiagen Column
1) The arabinose induced cells were pelletted and resuspended in 1ml of buffer B.
2) The cells were incubated with agaitation for 1 hour at room temperature.
3) The cell lysate was centrifuged at lO^OOOxg for 20-30 minutes at room temperature to pellet down cellular debris. The supernatant was collected.
4) The Ni-NTA column was pre equilibrated with 600)11 of Buffer B and centrifuged for 2 minutes at 7000xg.
5) Up to 600|-il of cleared lysate supernatant containing the 6X His tagged unprocessed peptide was loaded onto the pre-equilibrated Ni-NTA spin column. It was centrifuged for 2 minutes at 7000xg and the flow through was collected.
6) Ni-NTA column was washed with 600 |il of buffer C, centrifuged for 2 minutes at 7000xg and the eluate was collected.
7) The eluates were separated on 20 % Tris/Tricine Polyacrylamide gel.

8) To 100|il of unprocessed peptide purified by Ni-NTA column, lOjal of buffer (IM Tris pH 7.7) was added.
9) 1 |j.l [50ng] of the Endoproteinase Arg-C was added.
10) Incubated for 5hrs at 37 °C.
Spectrophotometric Assay for Antimicrobial Activities [figure 1]
1) 10^ cells were inoculated in 2X - TY medium
Tryptone : 16g/li
Yeast extract : lOg/li
Sodium chloride : 5g/li
2) The cells were allowed to grow till mid-log phase.
3) lOOfil of bacterial suspension was mixed with 100|il peptide suspension and mixed. Another aliquote of 100|il bacterial suspension was mixed with lOOjil of plain culture medium and this served as control.
4) Inhibition of proliferation was determined by OD measurements (620nm) after incubation period of 1 to 4 hours.
HEMOLYTIC ASSAY
1) Erythrocytes were isolated from heparinized blood by centrifugation .
2) Cells were washed thrice with Phosphate buffer saline (150mM KCl, 5mM Tris HClpH7.4,0.15MNaCl).
3) Erythrocytes were suspended in 10 volumes of PB S buffer (stock cell suspension).
4) The stock cell suspension was diluted 25 fold with PBS buffer and incubated in the water bath at 37°C for 15 min.
5) The test sample was added with antimicrobial peptide and incubated for Ih.
6) The samples were centrifuged at 4000xg for 5 min and the absorbance of supernatant was determined at 540nin.

We claim:
1. a process of bioproduction of antimicrobial peptide which has the amino acid
sequence ALWKNMLKALAKIVAR (single letter codes of amino acids) exhibiting antimicrobial activity without hemolytic activity, the said process comprising the steps of:
a) making a construct of nucleotide sequence encoding 17 aminoacid peptide
of the sequence " Argine - Alanine - Leucine - Trypto phan - Lysine -
Asparagine - Methionine - Leucine - Lysine - Alanine - Leucine - Alanine -
Lysine - Iso leucine - Valine - Alanine - Arginine",
b) cloning the coding fragment in frame in pBAD g IIIA plasmid,
c) expression of the cloned fragment by cultivating the transformed E, coli in
arabinose-containing medium,
d) purification of the said peptide using Ni-NTA affinity column and
e) cleaving the purified peptide using clostripain to give a peptide sequence of Alanine - Leucine - Tryptophan - Lysine - Asparagine - Methionine -Leucine - Lysine - Alanine - Leucine - Alanine - Lysine - Iso leucine -Valine - Alanine - Arginine.

Documents:

426-mas-2003-abstract.pdf

426-mas-2003-claims duplicate.pdf

426-mas-2003-claims original.pdf

426-mas-2003-correspondnece-others.pdf

426-mas-2003-correspondnece-po.pdf

426-mas-2003-description(complete) duplicate.pdf

426-mas-2003-description(complete) original.pdf

426-mas-2003-drawings.pdf

426-mas-2003-form 1.pdf

426-mas-2003-form 19.pdf

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Patent Number

197940

Indian Patent Application Number

426/MAS/2003

PG Journal Number

30/2009

Publication Date

24-Jul-2009

Grant Date

Date of Filing

22-May-2003

Name of Patentee

SAMBANDAM SHANMUGASUNDARAM

Applicant Address

DEPT, OF MICROBIAL TECHNOLOGY, MADURAI KAMARAJ UNIVERSITY, MADURAI 625021

Inventors:

#	Inventor's Name	Inventor's Address
1	SAMBANDAM SHANMUGASUNDARAM	DEPT, OF MICROBIAL TECHNOLOGY, MADURAI KAMARAJ UNIVERSITY, MADURAI 625021
2	Mrs, SRI LAKSHMI SEELAM	SCHOOL OF BIOLOGICAL SCIENCES MADURAI KAMARAJ UNIVERSITY MADURAI 625021
3	Dr, SUGUNA SHNMUGASUNDARAM	MADURAI KAMARAJ UNIVERSITY MADURAI 625021

PCT International Classification Number

A61K38/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA