Gene Synthesis and Fusion Expression D-Aminoacylase Gene from Alcaligenes A-6†

doi:10.7503/cjcu20140074

Abstract

Abstract:

The codons of D-ANase gene from Alcaligenes A-6 were substituted by the codons abundant in E. coli., then the D-ANase gene was synthesized by the two-step method based on PCR technology. Synthetic gene and pET-32a vector were digested with BglⅡ and XhoⅠ, ligated by T4 DNA ligase. The ligation mixture transformed into E.coli BL21(DE3) competent cell. Recombinant protein was detected by SDS-PAGE, Western-blot and activity assay. D-ANase can be expressed efficiently in E. coli and the expressed protein content can reach to 69.2% of the total bacterial protein content. In addition, the fermentation activity can achieve 96 U/mL. After the ultrasonic cell disruption, the recombinant protein was purified by Ni²⁺ affinity chromatography column. The specific activity of the purified recombinant enzyme was 1692.3 U/mg and the purity could be up to 95%. Furthermore a firm foundation was laid for the industrial use of the D-ANase.

Key words: D-Aminoacylase, Alcaligenes A-6, Gene synthesis, Fusion expression

CLC Number:

O629.74
Q786

TrendMD:

HOU Xintong, DONG Yuan, LIN Ruidong, YU Liang, REN Yuanyuan, LI Jianguang, GAO Chaohui, TENG Lirong. Gene Synthesis and Fusion Expression D-Aminoacylase Gene from Alcaligenes A-6^†[J]. Chem. J. Chinese Universities, 2014, 35(8): 1670.

Figures/Tables 5

Table 1 Condon usage of D-aminoacylase gene

Amino acid	Number of synonymous codons	Codon usage	Number of non- replaced codons	Amino acid	Number of synonymous codons	Codon usage	Number of non- replaced codons
Ala	66	GCT	0	Met	14	ATG	14
Cys	4	TGC	4	Asn	7	AAC	5
Asp	35	GAC	31	Pro	31	CCG	18
Glu	26	GAA	10	Gln	14	CAG	12
Phe	17	TTC	17	Arg	36	CGT	2
Gly	44	GGT	0	Ser	22	TCT	1
His	16	CAC	11	Thr	31	ACC	22
Ile	22	ATC	21	Val	33	GTT	0
Lys	7	AAA	1	Trp	4	TGG	4
Leu	43	CTG	36	Tyr	12	TAC	10

Fig.1 Agarose gel electrophoresis showing the synthesis result of DA-PCR(A) and OE-PCR(B) (A) Lane 1: 100 bp DNA marker, lanes 2—9: assembled by 6 single-stranded oligonucleotides, lane 10: assembled by 4 single-stranded oligonucleotides; (B) lane 1: 1000 bp DNA marker, lanes 2 and 3: full-length dan gene.

Fig.2 Agarose gel electrophoresis showing the recombinant PCR identification results Lane 1: 1000 bp DNA marker; lanes 2—7: identification of recombinant plasmid pET-dan.

Fig.3 SDS-PAGE analysis of expression and purified recombinant protein D-ANase Lane 1: protein marker; lane 2: pET-dan induced by IPTG in 0 h; lane 3: pET-dan induced by IPTG in 3 h; lane 4: the uncrushed cells of ultrasonic; lane 5: the pellet fractions of the induced; lane 6: the column effluent of Ni2+ affinity chromatography; lane 7: the eluted fractions by 50 mmol/L imidazole; lane 8: the eluted fractions by 300 mmol/L imidazole.

Fig.4 Western-blot analysis of recombinant protein D-ANase Lane 1: vector plasmid pET-32a induced by IPTG in 3 h; lane 2: recombinant plasmid pET-dan induced by IPTG in 0 h; lane 3: recombinant plasmid pET-dan induced by IPTG in 3 h; lane 4: purified recombinant protein D-ANase.

References 18

[1]	Yang H. K., Xu W. F., Duan A. N., You W. W., Zhao P. L., Chem. J. Chinese Universities, 2014, 35(3), 555—563
	(杨海葵, 许万福, 段安娜, 游文玮, 赵培亮. 高等学校化学学报, 2014, 35(3), 555—563)
[2]	Wang W., Xi H. G., Bi Q. R., Hu Y., Zhang Y., Ni M. X., Microbiol. Res., 2013, 186(6), 360—366
[3]	Liu J., Asano Y., Ikoma K., Yamashita S., Hirose Y., Shimoyama T., Takahashi S., Nakayama T., Nishino T., J. Biosci. Bioeng., 2012, 114(4), 391—397
[4]	Wakayama M., Yoshimune K., Hirose Y., Moriguchi M., J. Mol. Catal. B: Enzym., 2003, 23(2—6), 71—85
[5]	Tsai Y. C., Tseng C. P., Hsiao K. M., Chen L. Y., Appl. Environ. Microbiol., 1988, 54(4), 984—989
[6]	Moriguchi M., Ideta K., Appl. Environ. Microbiol., 1988, 54(11), 2767—2770
[7]	Moriguchi M., Sakai K., Katsuno Y., Maki T., Wakayama M., Biosci. Biotechnol. Biochem., 1993, 57(7), 1145—1148
[8]	Wakayama M., Ashika T., Miyamoto Y., Yoshikawa T., Sonoda Y., Sakai K., Moriguchi M., J. Biochem., 1995, 118(1), 204—209
[9]	Wakayama M., Katsuno Y., Hayashi S., Miyamoto Y., Sakai K., Moriguchi M., Biosci. Biotechnol. Biochem., 1995, 59(11), 2115—2119
[10]	Wakayama M., Hayashi S., Yatsuda Y., Katsumo Y., Sakai K., Moriguchi M., Protein. Expres. Purif., 1996, 7(4), 395—399
[11]	Yoshimune K., Ninomiya Y., Wakayama M., Moriguchi M., J. Ind. Microbiol. Biotechnol., 2004, 31(9), 421—426
[12]	Richardson S. M., Wheelan S. J., Yarrington R. M., Boeke J. D., Genome Res., 2006, 16(4), 550—556
[13]	Young L., Dong Q., Nucleic Acids Res., 2004, 32(7), e59-1—e59-6
[14]	An D., Zhao X. H., Zhou M., Ye Z. W., Chem. J. Chinese Universities, 2014, 35(2), 275—280
	(安东, 赵晓辉, 周密, 叶志文. 高等学校化学学报, 2014, 35(2), 275—280)
[15]	Gao C. H., Shi X. Y., Hou X. T., Meng Q. F., Zhang Y. J., Teng L. R., Chem. Res. Chinese Universities, 2008, 24(4), 487—490
[16]	Huang Z. G., Lin K. J., Yin H. P., Ye B. P., Weng X. B., You Q. D., Chem. J. Chinese Universities, 2013, 34(8), 1887—1893
	(黄振桂, 林克江, 尹鸿萍, 叶波平, 翁幸鐾, 尤启冬. 高等学校化学学报, 2013, 34(8),, 1887—1893)
[17]	Sa Z. P., Liu Y. J., Li Y. P., Zhu M., Li Y. X., Wang J. Y., Chem. J. Chinese Universities, 2013, 34(7), 1770—1775
	(撒宗朋, 刘彦晶, 李亚鹏, 祝明, 李玉祥, 王静媛. 高等学校化学学报, 2013, 34(7), 1770—1775)
[18]	Nakamura Y.,