Patent Application
20080138858
The present invention is related to the cloning of human arginase I. In particular, the present invention is related to nucleic acid molecules and plasmids that correspond to said human arginase I. The present invention also relates to a strain of E. coli for expression of said recombinant protein of human arginase I. The present invention also relates to a method of producing a recombinant protein.
Recombinant process uses genetically engineered organisms to produce useful proteins for medical use. Some examples of product made by recombinant process are insulin, growth hormones and vaccines. Large amounts of the protein can be produced in a factory with vats of the genetically engineered bacteria. In recombinant process, organism most commonly used is Escherichia coli.
Bacteria physiology and genetics are probably far better understood than for any other living organism. However, the success or failure of a process often depends on the survival rate of the genetically engineered bacteria and the recombinant DNA which carries the essential information for making the final product. Poorly constructed plasmid may become unable to produce meaningful amount of product yet lower the survival rate of the genetically engineered bacteria. There are also risks of producing contaminations hard to eliminate and worsen the quality of the final product.
In view of the foregoing background, it is an object of the present invention to provide a better genetically engineered bacteria in producing human arginase I so as to maximize output of producing said arginase, making the method safe and efficient for the production of pharmaceutical GMP grade material.
Accordingly, the present invention, in one aspect, is an isolated and purified nucleic acid molecule for the expression of recombinant human arginase I.
A preferred embodiment of the present invention is the use of the aforesaid nucleic acid molecule in constructing a plasmid for expression of recombinant human arginase I.
A further aspect of the invention is the use of the aforesaid plasmid in constructing an isolated strain of Escherichia coli for the production of recombinant human arginase I.
The plasmid pET30a(+)/ARGC plasmid was prepared using experimental techniques common in the field of gene cloning. First, both pAED-4/ARGC plasmid and pET30a(+) plasmid were independently subjected to overnight digestion at 37° C. with the restrictive enzymes NdeI and XhoI. The digested fragments were then mixed with T4 DNA ligase at 16° C. overnight. The ligated plasmid was transformed into competent DH5(α) E. coli cells. Selection was performed on LB plates comprising 30 μg/mL kanamycin. Single colonies were picked and cultured. The ligated plasmid was extracted and confirmed by digestion using the restrictive enzymes NdeI and XhoI at 37° C. for 1 hour and electrophoresis. Ultimately, the ligated and extracted plasmid contained a pET30(+) backbone and the human arginase gene (containing non-coding sequence) was named pET30(+)/ARGC. The nucleic acid sequence was confirmed by Invitrogen Biotechnology Co., Ltd (Shanghai). As shown in
The constructed pET30a(+)/ARGC was used to transform competent BL21 (DE3) E. coli cells on LB plates containing 30 μg/mL kanamycin. After 12 hours growth time, single colonies were picked and transferred into 50 mL LB media. The cells were fermented at 37° C. at 250 rpm. At OD600 0.6 to 0.8, IPTG was added to a concentration of 0.4 mM to induce expression. SDS-PAGE is used to test the expression level.
Two primers (SEQ ID NO. 1 and 2) were designed for the construction of pET30a(+)/ARGM plasmid using the restrictive enzymes NdeI and XhoI, as follows:
The plasmid pET30a(+)/ARGM was prepared using experimental techniques common in the field of gene cloning. First, amplify pAED-4/ARGC plasmid by Polymerase Chain Reaction (PCR) using pAED-4/ARGC plasmid as the template. The amplified gene fragments and pET30a(+) plasmid were independently subjected to overnight digestion at 37° C. with the restrictive enzymes NdeI and XhoI. The digested fragments were then mixed with T4 DNA ligase at 16° C. overnight. The ligated plasmid was transformed into competent DH5(α) E. coli cells. Selection was performed on LB plates comprising 30 μg/mL kanamycin. Single colonies were picked and cultured. The ligated plasmid was extracted and confirmed by digestion using the restrictive enzymes NdeI and XhoI at 37° C. for 1 hour and electrophoresis. Ultimately, the ligated and extracted plasmid contained a pET30(+) backbone and the human arginase gene (without the non-coding sequence), was named pET30(a)/ARGM. The nucleic acid sequence was sent to and confirmed by Invitrogen Biotechnology Co., Ltd (Shanghai). As shown in
The constructed pET30a(+)/ARGM was used to transform competent BL21 (DE3) E. coli cells on LB plates containing 30 μg/mL kanamycin. After 12 hours growth time, single colonies were picked and transferred into 50 mL LB media. The cells were fermented at 37° C. at 250 rpm. At OD600 0.6 to 0.8, IPTG was added to a concentration of 0.4 mM to induce expression. SDS-PAGE is used to test the expression level.
Table 1, 2 and 3 show the comparison of physiological characteristics of E. coli cells transformed with pAED-4/ARGC, pET30a(+)/ARGC and pET30a(+)/ARGM, in terms of plasmid stability. Initially, E. coli cells transformed with pAED-4/ARGC and pET30a(+)/ARGC showed normal growth rate and kanamycin resistance. After 4 months of storage in glycerol at −80° C., no colony was detected until the dilution fold was decreased to 10e4-10e5, and no gene expression was detected from the fermentation broth.
E. coli cells transformed with pET30a(+)/ARGM initially showed normal kanamycin resistance at the dilution fold of 10e9-10e10. Also, expression level was found to be 15% to 25%, which was much higher than that of pAED-4/ARGC and pET30a(+)/ARGC transformed cells. After 6 months of storage in glycerol at −80° C., pET30a(+)/ARGM transformed cells retained the normal level of kanamycin resistance, and expression level was much higher than that of pAED-4/ARGC and pET30a(+)/ARGC transformed cells after 4 months −80° C. storage.
The preferred embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details. Hence, this invention should not be construed as limited to the embodiments set forth herein.
For example, although the present invention referred to using pET30a(+) vector from Novagen, a person skilled in the art will appreciate that other vectors may be employed, such as pTrcHis (Invitrogen), pGEX (Amersham Biosciences), pBAD (Invitrogen), pRSET (Invitrogen), pBV220, and pQE (Qiagen).
A person skilled in the art will also appreciate that although the present invention referred to using a lac promoter, a person skilled in the art will appreciate that other promoters may be used, such as tryptophan promoter, Trc promoter, Tac promoter, araBAD promoter, T7 promoter, T5 promoter, and temperature induced promoter.
Furthermore, a person skilled in the art will also appreciate that although the present invention referred to using BL21(DE3) as host, other expression systems may be employed, such as TOP10, M15, and DH5a E. coli.
The present invention has been described using the encoding region of human arginase I, which consists of 990 bp including the final TAA which transcribes into the stop codon UAA. The most preferred embodiment of the present invention uses an encoding region of human arginase I consisting of 993 bp, which an additional set of TAA is included to further ensure the expression of the terminal signal.
