Design of new primer for expression of microbial L-asparaginase as anticancer agent using genetic engineering and bioinformatics applications.
Mohammed Mahmoud shawky Kassab,molecular
biologist and specialist of pharmacology,toxicology
microbiology and immunology,faculty of
pharmacy,Cairo university,Egypt.
Abstract
L-asparaginase is an ideal enzyme in the treatment of lymphoblastic leukaemia.It is also effective in food industry because it decreases acryl amide formation which is a potent carcinogenic metabolite.L- asparaginase was produced from different microbial sources such as Escherchia coli and Erwinia chrysanthem.In our study,we designed new primer for expression of microbial L-asparaginase as anticancer agent.This primer was characterized with pure and high yield of the enzyme of interest.We used some genetic engineering and bioinformatics applications for designing this primer.using Escherchia coli as bacterial vector for cloning and expression and PCR based methods ,we could obtain higher yield of therapeutic enzyme of interest (generated from our new microbial primer for expression of L-asparaginase enzyme)than standard bacterial L-asparaginase produced from Escherchia coli .We recommend other researchers to try to design new primers for L- asparaginase from fungal sources because it possess less side effects than bacterial one.This is because fungi are eukaryotic which is similar to eukaryotic human cells.
Introduction
Cancer chemotherapy:
Cancer diseases such as acute lymphocytic leukaemia ,testicular cancer are treated with anticancer drugs which produce high cure rates(1). High mortality rates occur without chemotherapy for cancer diseases(2).The anticancer drugs are the most toxic pharmacologic drugs(3).A lot of anticancer drugs are cytotoxic agents that act on dividing cells(4) .Anticancer drugs include alkylating agents (eg,cisplatin and cyclophosphamide),antimetabolites(eg,5-fluorouracil,methotrexate, gemcitabine and 6-mercaptopurine),miscellaneous agents(eg,imatinib,L- asparaginase and cetuximab),hormonal agents(eg,tamoxifen and prednisone),anticancer antibiotics(eg,bleomycin,doxorubicin and
2
mitomycin) and natural products(eg,etoposide,vincristine,vinblastine and paclitaxel)(5).
Cell cycle specific agent:
An anticancer agent that acts selectively on tumor stem cells when they are traversing the cell cycle and not when they are in G0(resting) phase(6).
Cell cycle nonspecific agent:
An anticancer agent that acts on tumor stem cells when they are traversing the cell cycle and when they are in G0 phase(7) .
Oncogene:
A mutant form of a normal gene that is found in naturally occuring tumors and which when expressed in noncancerous cells,causes them to behave like cancer cells(8).
Growth fraction:
The proportion of cells in a tumor population that are actively dividing(9).
Pharmacology of L-asparaginase:
It is used in treatment T-cell auxotrophic cancers(lymphomas and leukaemia) that need exogenous asparagine which is necessary for growth(10).Depletion of serum asparagine is caused by asparaginase(11).Asparaginase is given by intravenous route(12).It may cause bleeding ,acute pancreatitis and hypersensitivity reactions(13).
Production of microbial L-asparaginase:
It is produced from prokaryotes as Escherchia coli and eukaryotes as some fungi(14).The aim of our study is to produce pure and high amounts of microbial L-asparaginase through genetic engineering and bioinformatics using new primer for cloning and expression design.
Methodology
1- Copy the fasta of microbial L-asparaginase genome from NCBI.
2- Copy coding sequence(CDS)of microbial L- asparaginase enzyme from NCBI.
3- Choose the vector plasmid such as PUC18.
4- Choose suitable restriction enzymes for forward and reverse primers according to webcutter website.
Criteria for choosing suitable restriction enzyme
A-Enzyme should be in multiple cloning site(MCS) of plasmid vector. B-Available
C-Obey orientation of gene of interest to be cloned and expressed.
After determining suitable restriction enzymes ,forward and reverse enzymes, the T annealing for forward and reverse primers are determined using PDRAW32 microsoft application.Difference between annealing tempreature of forward and reverse primers should not exceed 3
3
centigrades.This is optimal condition for cloning and expression of the protein of interest.
5- Isolation or synthesis of nucleotide sequence of L-asparaginase enzyme.
6- Insertion of this sequence in a suitable expression vector such as PUC18. the inducible IPTG is added.
7- Transformation of a suitable host cell such as saccharomyces cerevisiae(which is characterized by expression of extracellular proteins) by the prepared recombinant DNA.
8- Selection of the right clone .
9- Establishment of process of production of interested enzyme.
10- Isolation of extracellular L-asparaginase enzyme from the supernatant of centrifuge tube after centrifugation by crystallization with ammonium sulfate.
11- Determine the activity of enzyme by direct nesslerization test and measuring the absorbance at 420 nm in presence of asparagine amino acid as the substrate for enzyme.
Results and discussion
Fasta of microbe producing L- asparaginase genome
AAGCTTTGTGGCATTGCTGCAACAAACGCACTTGAGTTGG GGATTGATGTTGGTCACATCGATGTAACTC TTCATTTAGGTTTTCCCGGGAGTATTGCCAGGTAAATGAGT GTGATTTACTCTTTTCTTTTCCCTCTTAG
AACTGAAATTCTATAAGCTGCCGCTGACAATGACTGATCAA TTTTGAGTTTCAGTCTTTGGCAGCAAGCC GGTCGGTCTGGAAGGCGACAAAAGCCCTCTCTTGCTGTAT ATGTTGCCTTTCTCGGCCCGCTTGACCAAT ACTACATGAGCTTTCCGGACAAACTCTTTGGTAGCCCAATT GAGTGTTGCCATATTGATTCTCAAAATAA GCATGTTAGTAGTATATACTGTTTGGGTTCTTGTGCCAACT GTTAATACTTAATACTCTAGCTGAGATTA TACCAACTTGCTTTGATACTTTTTCTTCTTGTAGGTCCTTAT GCAACATTTGGCTTGTGCTGCTCTTGAG
CATCCGTTGAGCTTACAATATGATCAACAGCACTTTGGTTC TGGCTTAAGCGACCCTCTTGCTGAGCTCA GGAACAAAGGATTCTTGAGTTTTGATCCATCCCGTGATTCT TCTTCTAGAATATGGAACTATATTGGTCG AGAGGTATATATCCGAGCAGTTACTTTGAAAAATACAACCC GAAACCGAGTTTCCTTGCTCTAAATACCT
TTGCTGGTCAACATTTTCTCAGAAAAATCCTGCGAGGATAG TCTCTATACGGGCCATAGAGACAGTAAGA
4
TACAGAGTCATGGAAAAAAAAAGTAAAGATGTTCTCGATGA AATCGAGGAAAAGCAAAGCGTTTTTCCAC GTATATGAAGGAGCTATTTATATGAACCAAGGAAGGAACTA CCTGGTCACGAGTCTGGATATAAAAGAAA AGGTAGCTCTGTGTGAACTAGTAAACGTGGATTACTACACC AGGACTCGGGATTACACTGATATCAAAGT AACTGGTGGTGACACTGTAAGTATAACTGTGGCTCTCTTGT TTGATAGTTGTGCATCTTTGTGTAAAATT CATTACCAAGTTCATGTTTCAGGCTTACCCGGTCAAGGCTC
CAAAGAAGCCAACACCACAAACTCATGCA TGTAGAGTGACAACAAAGTGGTTTGGATTTCTTCGTATCCG AAGAAGAAACAATGAAGTAATTGATGATG TTGAGCTTTCACTCCCAAGTTACACTTACCAATCACAGGTT ATATTCTTAAACATGCAGCCTCACACAAC ACACATACAGTCTTTTCTGTTTCCGCTGTAATTTTAAGTCT GGTTTTATTGTGGATAATAGGCTGTTTGG ATTCAAGTGCCGATGTCGGTAAAACTAGCAGTGGAGACGG CAAACTTGCCATTCCGTGCTGGTTTACACG CAGCTTGTCACGCCCTTGTCAACGTTGTTCCAACAAGAGTG ACATGCAACTATTCAGACATAGCTCCAGA ATGTCCAAATCCGCAAGAGCAACGGTATTTCCCAGCAAGA ATCTTAGTATATGATCGTCATCCTGGAGGA ACTGGCATATCCGCCAAGGTAAGTGGAAGTAAAACTGTGA AAGTAAATTCTCAAGTTTCAATTGTTGAAT
ATGGATGACTAATTCTTGAACATTTTGTTCTGCAGATCTGT CCGCTCTTCTTTGAGCTTCTAAAAGATGC TCGTGATCTCTTAAGGTCTTGCGAAAAATGCCCACCTGAAA CTGGTTGCCCCAAGTGCGCTCAGACTTTT GGTTGCCGAGGGTACAATGAACTCCTACATAAGAAAGCAG CCATCATGATCATGCAGGTAACCAAAAAAA ATACTAAATGATTTCTTACTTTTTTTCTATATTACTACAAAA ACTTTTAAAAAGAGATTGGCTATAAAGC CTGATCATTGTTATCTAATATTGCGTTTAGGGCGTTTTGGA CGCGAAGGAAGATTGAATCATCAGACATC
TCAAGATTACAATTTTTTTCATTTTTTGTAAATCTAATTCAT TCAAAAGGTTAAAATAGATTTGATTGAC TGACTAGTTAACAACACATTTTTGTTACTAATAAAAATCTT GTAATTGACACACACACAAAAAAAAAAAA AACTTGTAATTCAATAATAATATAAAGTTTGAATCTGAAAA GATCTGTAACTAAAAGGCTTTAAAATAGT
CTGGGAAAAGTGAGCCAACGTGTCTTTAAGTTATACGGCG GAAGATGGTGGGGTGGGCGATTGCGCTACA
5
CGGCGGTGCCGGAGACATTCCGATCGATCTCCCCGACGAG CGACGTATCCCTCGTGAGAGCGCCCTCCGT CACTGCCTCGATCTTGGCATCTCCGCCCTCAAATCCGGCAA GCCTCCCTTGGACGTCGCCGAACTTGTCG TACGCCTCTCTTTCTCGATAATTGTTTTTTTTTCTTTGGCAT ATGGCAGCTGGTCGGATCCATGCGTTTT GATAATTTTGTCTAGAGTTTGGAATAATCGATGACGAAACA GAGAATTCGGAACCAATTTTAGATTTATT TTTAAAACTCACTCTCTCTAGTGATCAGTTAACTAGTCCCA
TATTTGATACGATCACCATTTGTAAGGTT CGTGAACTTGAGAACCACCCGGACTTCAATGCGGGTAAAG GATCTGTCTTAACTGCACAAGGCACTGTTG AAATGGAAGCTTCCATTATGGACGGTAAAACCAAAAGATGT GGAGCTGTCTCCGGCTTGACCACTGTTGT TAATCCCATTTCTTTAGCTCGCCTCGTCATGGAGAAAACTC CTCATATATATCTTGCATTCGATGCTGCT GAAGCTTTTGCAAGAGCACATGTCAGTGTATTCTACTCTCT TCTTTTGGTGACTTGTATTGATTATTAAA CTCTGCTCATTGTAGATCTTTCCATGGATTCCGGTTTTATA GGGTGTTGAGACGGTATATTCTAGCCATT TCATAACTCCTGAAAACATTGCAAGGCTAAAGCAGGCCAAA GAATTCAATCGAGTCCAGGTACTAAGTTT TTTCTCGAGGTTTGTGTTCATCAAATGATGATATAATCCTC CCCTTGCCTTGTGCTTCTTAACGATCTTT
AAATTCCTTGATTACAGTTGGATTACACAGTCCCTAGTCCG AAAGTACCGGACAATTGCGGTGACAGCCA AATAGGAACGGTCGGATGTGTAGCTGTGGACAGTGCTGGA AATCTAGCTTCGGCTACATCAACGGGCGGT TATGTCAACAAAATGGTTGGCAGAATTGGGGATACGCCAG TCATTGGCGCAGGAACTTACGCTAACCACC TTTGTGCCATCTCAGCCACAGGTAAAGGAGAGGATATCATC CGTGGAACCGTGGCTAGAGACGTGGCTGC ACTCATGGAATATAAAGGCTTGTCTTTGACTGAGGCAGCG GCTTATGTTGTTGACCAATCTGTTCCCAGA
GGAAGCTGTGGACTCGTTGCTGTCTCTGCCAATGGTGAAG TCACAATGCCGTTTAACACTACCGGAATGT TCAGGGCTTGTGCTAGCGAAGATGGTTACTCTGAGATCGC AATCTGGCCAAACAATTGAAATCACTAGAC GGAGAGGAATGGCTTTGTCTATTATTAAGCAACTCTGTTTA ATCTTCTTCTTTAGTTTCAGTTTGTAAGT
TGTAACCTTATATGACTCAGTCTTGCAGTATTTTAGTTCTTC GGATTTGGTTTGATGATTTCGGTCTAGG
CDS of microbial L- asparaginase :-
Forward primer for expression microbial L-asparaginase:-
GTCCCTAGTCCGAAAGTACCGGACAATTGCGGTG
Reverse primer for expression of microbial L-asparaginase:-
GGGTTTTCGGATAAAAAATTGTTAGAAAAAAAC
1- 2-
6
ATTGTTCGGTTTGGAATGATGGGTTTTCGGATAAAAAATTG TTAGAAAAAAACAAGCAAAGTAACAATCA CTGTTTGTTTAATGGTTCTA
ATGGTGGGGTGGGCGATTGCGCTACACGGCGGTGCCGGAG ACATTCCGATCGATCTCCCCGACGAGCGAC GTATCCCTCGTGAGAGCGCCCTCCGTCACTGCCTCGATCTT GGCATCTCCGCCCTCAAATCCGGCAAGCC TCCCTTGGACGTCGCCGAACTTGTCGTTCGTGAACTTGAGA
ACCACCCGGACTTCAATGCGGGTAAAGGA TCTGTCTTAACTGCACAAGGCACTGTTGAAATGGAAGCTTC CATTATGGACGGTAAAACCAAAAGATGTG GAGCTGTCTCCGGCTTGACCACTGTTGTTAATCCCATTTCT TTAGCTCGCCTCGTCATGGAGAAAACTCC TCATATATATCTTGCATTCGATGCTGCTGAAGCTTTTGCAA GAGCACATGGTGTTGAGACGGTATATTCT AGCCATTTCATAACTCCTGAAAACATTGCAAGGCTAAAGCA GGCCAAAGAATTCAATCGAGTCCAGTTGG ATTACACAGTCCCTAGTCCGAAAGTACCGGACAATTGCGGT GACAGCCAAATAGGAACGGTCGGATGTGT AGCTGTGGACAGTGCTGGAAATCTAGCTTCGGCTACATCA ACGGGCGGTTATGTCAACAAAATGGTTGGC AGAATTGGGGATACGCCAGTCATTGGCGCAGGAACTTACG CTAACCACCTTTGTGCCATCTCAGCCACAG
GTAAAGGAGAGGATATCATCCGTGGAACCGTGGCTAGAGA CGTGGCTGCACTCATGGAATATAAAGGCTT GTCTTTGACTGAGGCAGCGGCTTATGTTGTTGACCAATCTG TTCCCAGAGGAAGCTGTGGACTCGTTGCT GTCTCTGCCAATGGTGAAGTCACAATGCCGTTTAACACTAC CGGAATGTTCAGGGCTTGTGCTAGCGAAG ATGGTTACTCTGAGATCGCAATCTGGCCAAACAATTGA
The puc 18 plasmid was used as the the vector
The two restriction enzymes that were used and chosen according to
webcutter website were:-
Bam HI.
Sphl.
In our study to determine the annealing temperature we applied pdraw32
software application.
T.
annealing=TM-5=59-5=54.
7
Determination of activity of L-aspraginase enzyme by direct
nesslerization test:
This test revealed that the test L-asparginase catalyzed the conversion of
L-asparagine into aspartic acid and ammonia.the ammonia concentration
was determined spectrophotometerically by measuring the absorbance at
420 nm.The concentration and activity of L.asparaginase was directly
proportional to ammonia concentration.
The purity and yield of the produced test enzyme:
There was high yield and purity of test enzyme which was compared with
standard L-asparaginase enzyme.
Conclusion
The designed new primer for microbial L-asparaginase produced better
efficacy and higher yield of enzyme than commercial L-asparaginase
present in the pharmaceutical market.It is recommended that in future
studies to design new primers from fungal sources which are eukaryotic
organisms thus minimizing hypersensitivity reactions and obtaining
higher yield of L-asparaginase enzyme.
