PRODUCTION OF FULL LENGTH AND SPLICING FORM OF CHYMOSIN USING PETEXPRESSION SYSTEM IN E-COLI AND INVESTIGATION ITS ENZYME ACTIVITY AND PREPLASMIC SECRETION

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AHMADI ZEYDABADI M.; AHMADIAN GH.R.; SOROURI R.

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Journal: Koomesh Year:2008 | Volume:9 | Issue:3 (27) Page(s): 187-194

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Title

PRODUCTION OF FULL LENGTH AND SPLICING FORM OF CHYMOSIN USING PETEXPRESSION SYSTEM IN E-COLI AND INVESTIGATION ITS ENZYME ACTIVITY AND PREPLASMIC SECRETION

Author(s)

AHMADI ZEYDABADI M. | AHMADIAN GH.R. | SOROURI R. | Issue Writer Certificate

Keywords

PREPROCHYMOSINQ3

ASPARTYL PROTEINASEQ3

ALTERNATIVELY SPLICED TRANSCRIPTQ3

IN VITROQ3

E.COLIQ3

Abstract

Introduction: Chymosin (Rennin EC 3.4.23.4) is an aspartyl proteinas (the major proteolytic enzyme in the fourth stomach of the unweaned calf) that is formed by proteolytic activation from zymogene prochymosin. The aim of his study was to produce the full length and splicing form of chymosin using pETexpression system in E.COLI and to assay the activity of expressed enzyme and preplasmic secretion.Materials and Methods: The sense primer F-prochy(+) (5´-ggggccatgGCTGAGATCACCAGGA) including NCOI restriction site). The anti sense R-prochy(-) (5´- gggcggccgcGATGGCTTTGGCCAGC -3´) hybridizing to the C-terminal end of calf preprocymosin cDNA and contains an additional NotI restriction site at its 5´-end . The cells were disrupted by sonication and proteins were purified by using Ni-NTA beads from Qiagen under native conditional. The PREPROCHYMOSIN and AS6 PREPROCHYMOSIN were activated at pH 4.7. The enzyme solutions were diluted 20-fold with 50 mM phosphate buffer.Results: Sequencing data analysis showed that the exon six has been spliced out and, therefore the gene product is 114 bp shorter in length, both chymosin forms were expressed together in E.COLI. Under the same expression conditions, at least AS6 PREPROCHYMOSIN was produced 7-fold high expression in comparison to a full-length recombinant chymosin. Following acid activation and neutralization, the purified fractions were tested in a qualitative milk clotting assay. The clotting activity of PREPROCHYMOSIN and exon6-less PREPROCHYMOSIN were comparable.Conclusion: High expression of this alternatively expressed transcript in bacteria, and proper folding of the AS6 chymosin protein molecule in the absence of exon six are the two most important aspects distinguished in this research.

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APA: Copy

AHMADI ZEYDABADI, M., AHMADIAN, GH.R., & SOROURI, R.. (2008). PRODUCTION OF FULL LENGTH AND SPLICING FORM OF CHYMOSIN USING PETEXPRESSION SYSTEM IN E-COLI AND INVESTIGATION ITS ENZYME ACTIVITY AND PREPLASMIC SECRETION. KOOMESH, 9(3 (27)), 187-194. SID. https://sid.ir/paper/37381/en

Vancouver: Copy

AHMADI ZEYDABADI M., AHMADIAN GH.R., SOROURI R.. PRODUCTION OF FULL LENGTH AND SPLICING FORM OF CHYMOSIN USING PETEXPRESSION SYSTEM IN E-COLI AND INVESTIGATION ITS ENZYME ACTIVITY AND PREPLASMIC SECRETION. KOOMESH[Internet]. 2008;9(3 (27)):187-194. Available from: https://sid.ir/paper/37381/en

IEEE: Copy

M. AHMADI ZEYDABADI, GH.R. AHMADIAN, and R. SOROURI, “PRODUCTION OF FULL LENGTH AND SPLICING FORM OF CHYMOSIN USING PETEXPRESSION SYSTEM IN E-COLI AND INVESTIGATION ITS ENZYME ACTIVITY AND PREPLASMIC SECRETION,” KOOMESH, vol. 9, no. 3 (27), pp. 187–194, 2008, [Online]. Available: https://sid.ir/paper/37381/en

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