Cloning and In Vitro-Transcription of Chymosin Gene in E. coli~!2009-12-11~!2010-01-07~!2010-02-02~!

Abstract

Chymosin, commonly known as rennin, is the main milk-coagulating enzyme available in rennet. RNA was extracted from the abomasum of a suckling calf water buffalo and was subjected to RT-PCR using degenerate primers to amplify 850bp of the chymosin gene. The sequence was aligned with 19 different mammals' chymosin genes. The sequence revealed that there is a similarity to them ranging from 64% to 98%. The purified recombinant proteins were obtained from the transformed E. coli and yeast. The clotting activity of both of the resulting proteins was examined compared to the commercial peers. It was noticed that the concentration of the purified protein ranged from 15,000 to 40,000 MCU. Therefore, the activity of the obtained proteins was the same and it was 105% when compared to the commercial peer. Having examined the cytotoxicity of the purified proteins, the results revealed no toxicity. We can conclude that the obtained recombinant protein is more active and safe even when expressed in bacteria rather than yeast.

Cloning and Expression of Camel Pro-Chymosin Encoding Gene in E. coli and Characterization of the Obtained Active Enzyme

“…Recombinant pro-chymosin is expressed in bacteria in insoluble form and it is presented in the inclusion bodies. This finding has been reported for the production of recombinant bovine chymosin in E. coli (Mule et al, 2009;El-Sohaimy et al, 2010 andMenzella, 2011). The recombinant camel chymosin behaves like the bovine one that is presented in inclusion bodies and is not presented in the cell free extract (Fig.…”

“…Therefore, it could be concluded that biologically active chymosin expressed in fungi (Kappeler, 2006) or yeast (Wang et al, 2015) can be produced in E. coli after solubilization, renaturation, and acidic activation. Although, calf chymosin has been expressed in E. coli and it was active after solubilization, renaturation, and acidic treatment (El-Sohaimy et al, 2010 andMenzella, 2011). Optimal pH for the produced recombinant camel chymosin:…”

“…So, biologists solve this problem by recombinant producing mammalian chymosin to provide the enzyme requirement in cheese industries. Currently, recombinant chymosin is used for one-third of the cheese production over the world (Starovoitova et al, 2006 andEl-Sohaimy et al, 2010).…”

“…The calf chymosin gene was among the first mammalian genes that were cloned in different microorganism. Using recombinant DNA technology, calf chymosin encoding gene was expressed in GRAS (Generally Recognized As Safe) microorganisms as bacteria (Nishimori et al, 1982;Emtage et al, 1983;El-Sohaimy et al, 2010;Menzella, 2011 andNoseda et al, 2016), yeast (Mohanty et al, 1999;Starovoitova et al, 2006;Zhang et al, 2009;Noseda et al, 2013;Wang et al, 2015 andNoseda et al, 2016), filamentous fungus (Cardoza et al, 2003and Sharma et al, 2009), and plant (van Rooijen et al, 2008.…”

“…Expression of the pro-chymosin in yeast or fungus resulting on production of the enzyme in an active form. While, expression of the pro-enzyme in E. coli exit as insoluble form which needs further solubilization and renaturation of the enzyme from the inclusion bodies (Mule et al, 2009 andEl-Sohaimy et al, 2010). The major benefit of this insoluble form that it makes purification and activation could be achieved since most of E. coli proteins (about 90%) are precipitated under the acidic condition which is used to activate the pro-chymosin (Rodríguez-Carmona, 2010).…”