- synthesis, cloning and expression in escherichia coli of a gene coding for Mcoti-ii. - MCoTI-II is the most abudant inhibitor, consisting of 34 amino acid residues with 3 disulfide bridges, resistant to cleavage by thermolysin for more than 48 hours at 50 0 C. - In this work, chemical synthesis, cloning and expression of the gene coding for MCoTI-II are presented.. - Expression and purification of recombinant MCoTI-II were followed the manufactures instruction of IMPACT-CN System, using chitin beads column chromatography [7].. - Design and construction of the gene coding for MCoTI-II. - The nucleotide sequence coding for MCoTI-II was reversely translated from its amino acid sequence (fig 1, fig 2).. - Fig 1: The amino acid sequence of MCoTI-II [5]. - Fig 2: The nucleotide sequence derived from MCoTI-II amino acid sequence. - While desingning the oligo nucleotide primers Nde I restricsion site was introduced on the 5’ end, stop codon and Xho I restriction site was introduced on the 5’ end of the proposed synthetic trypsin inhibitor gene. - The sequences of the forward and reversed primers are shown on fig 3 and of the synthetic gene is on fig 4.. - Fig 3: The sequences of the forward and reversed primers for MCoTI-II gene cttaaggtatactcgccgtcgcgtaccgccgcacacgggcttt. - Fig 4: The sequence of synthetic MCoTI-II gene. - Amplification of MCoTI-II gene by PCR. - MCoTI-II gene was also successfully prepared by using forward , reverse primers and the purified total MCo DNA as template. - 200µM of dNTPs ;400ng of the purified total MCo DNA. - Cloning of the synthetic MCoTI-II. - Fig 5: Analysis of MCoTI-II gene on 5%polyacrylamide gel. - Lane 2 : PCR product of MCoTI-II gene. - Fig 7: Scheme summarizing the steps in obtaining a construct system for expression of MCoTI-II. - Fig 8: Electrophoresis of the PCR product of MCoTI-II gene on 2% agarose gel (a) and estimation of its size by using SHARP JX-330 scanner (b). - Transformed into E.coli BL21 (DE3). - PI - 17 pTYB12 – MCoTI-II. - M: DNA Marker 100bp (80-1031bp)(#SMO241/2/3) 18: PCR products of MCoTI-II gene. - Sequence of recombinant plasmid DNA containing TI gene fragment Expression of recombinant MCoTI-II (ReMCoTI-II). - The growth and induction conditions of PI-17 giving maximal yield of the recombinant fusion protein established as follows : cells were grown in shaker flasks at 37 0 C in LB medium with ampicillin (100µg/ml), induction at the mid-log phase with 0.5mM IPTG at 15 0 C for 16h.. - As mentioned in the methods, cloning and expression procedures were followed the IMPACT-CN system and pTYB12 vector was used ,so it was expected to produce 55kD fusion of the cleavable intein tag to N-terminus of a synthetic inhibitor .As known, Mr of MCoTI-II is about 3.4kD, hence Mr of the obtained recombinant fusion protein should be about 58 kD ( the sum of 55 + 3.4kD of MCoTI-II). - Purification scheme of recombinant MCoTI-II following IMPACT TM - CN system using chitin beads column chromatography comprsed the following steps. - 5) Elution of the target protein by CB buffer.. - Fig 11: Purification scheme of recombinant MCoTI-II using chitin beads column chromatography. - Fig 12 showed chitin gel before (N1) and after (N2) releasing MCoTI-II.. - Re MCoTI-II. - ♦ The MCoTI-II gene was synthesized by four overlapping primers ,transformated into pTYB12 vector. - ♦E.coli BL21(DE3) strain was used as the host for cloning and expression of the recombinant gene. - ♦The recombinant MCoTI-II was firstly synthesized in a 58kD fusion protein, then released from it and purified following IMPACT-CN system manufactures instruction by using chitin beads column chromatography. - ♦ The obtained purified recombinant MCoTI-II showing inhibitory activity against trypsin,and proteinases from Plutela xylostella. - Solution Structure of the Macrocyclic Squash Trypsin Inhibitor MCoTI-II, the first member of a new family of cyclic Knottins. - The Wave of the Future, Michal Lebl and Richard A. - Markiewicz and M.Fkus, Synthesis, cloning and expression in E.coli of the gene coding for the Trypsin inhibitor from Cucurbita pepo, Acta Biochimica Polonica 42, N 0 1(1995), pp. - E., Zhang M-F., Chen C-Q., Chemical synthesis, molecular cloning and expression of the gene coding for the Trichosantes trypsin inhibitor. - Laemmli U.K.,Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature V.227 (1970) pp 680-685. - Leluk J., and Pham T.T.C., Zastosowanie edestyny do badania aktywnosc proteinaz i ich inhibitorow (Polish ) in XXI Meeting of Polish Biochemical Society Abstracts, pp.139 (1985), Krakow, Poland. - Annual Meeting of the American Society of Biochenistry and Molecular Biology, August 24-29, San Franciso, California, Program N 0 - 2130, 1997.