Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

doi:10.3969/j.issn.1004-1524.2021.01.05

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (1): 34-42.DOI: 10.3969/j.issn.1004-1524.2021.01.05

• Animal Science • Previous Articles Next Articles

Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

LIU Jinyu¹(), HUANG Ying²^,^*()

1. College of Life Science, Yangtze University, Jingzhou 434025, China
2. College of Life Sciences, Nanjing Normal University, Nanjing 210023, China

Received:2020-08-01 Online:2021-01-25 Published:2021-01-25
Contact: HUANG Ying

Abstract

Abstract:

To bioinformatically analyse the biological characteristics of Schizosaccharomyces pombe SpTrz2 protein, both the full-length and the N-terminal half of S. pombe sptrz2 gene were amplified by PCR and inserted into pET-28a(+) expression vector through NdeⅠ and XhoⅠ restriction enzyme sites. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) and the transformants were induced by IPTG. Then recombinant proteins were purified by the way of SDS-PAGE gel slices. Polyclonal antibodies were prepared by immunizing New Zealand white rabbit using the purified recombinant proteins. The specificity of antibodies was tested by Western blotting. The results showed that SpTrz2 was a transmembrane protein with three transmembrane domains and contained multiple B cell antigen epitopes. SDS-PAGE analysis suggested that the full-length and the N-terminal half of SpTrz2 protein (SpTrz2 and SpTrz2N) with a relative molecular mass of about 75.99 ku and 44.77 ku, respectively, mainly existed in a form of inclusion body. The Western blot analysis showed that polyclonal antibodies of SpTrz2 and SpTrz2N could specifically recognize natural SpTrz2 protein. In this study, recombinant SpTrz2 and SpTrz2N protein were successfully expressed and purified, and their polyclonal antibodies were prepared. All above results could provide experimental evidence for the study of biological function of SpTrz2 protein.

Key words: Schizosaccharomyces pombe, SpTrz2 protein, prokaryotic expression, polyclonal antibody

CLC Number:

Q939.9

LIU Jinyu, HUANG Ying. Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 34-42.

Figures/Tables 9

Table 1 PCR primers for the amplification of target gene fragments

引物名称 Primer name	引物序列(5'→3') Sequences(5'→3')	限制性内切酶 Restriction enzyme
trz2-fo	GGGAATTCCATATGAAAGCTTCTCTTCTGGTTCCA	NedⅠ
trz2-re	CCGCTCGAGTATCGTTTCTCGCTGCTTGC	XhoⅠ
trz2N-dw	CCGCTCGAGTCCTTCATTTTCAAAGGGTAAAGGA	XhoⅠ

Fig.1 Sequence alignment of the N-terminal halves of tRNase ZLs ScTrz1-N, the N-terminal halve of tRNase ZL protein from S. cerevisiae; SpTrz2-N, the N-terminal halve of SpTrz2 protein from S. pombe; DmTrz1-N, the N-terminal halve of tRNase ZL protein from D. melanogaster; ELAC2-N, the N-terminal halve of ELAC2 protein from Homo sapiens; Black lines indicated ψ motif II, flexible arm, GP and Walker A-like motifs; Identical residues that were conserved in at least 80% of the aligned sequences were shaded in black; Similar residues were shaded in grey; Numbers in parentheses were the positions of the amino acid sequences.

Fig.2 Transmembrane domain of SpTrz2 protein

Fig.3 B cell epitopes in SpTrz2 protein A,The B cell epitopes in the N-terminal halve of SpTrz2 protein; B, The B cell epitopes in the C-terminal halve of SpTrz2 protein.

Table 2 Amino acid sequence of B cell epitope in the N-terminal halve of SpTrz2 protein

编号 No.	开始 Start	结束 End	肽段序列 Peptide sequence	氨基酸长度 Length/aa
1	22	24	YSW	3
2	39	43	KSKRN	5
3	55	57	LNP	3
4	90	97	QASNYGGK	8
5	128	130	GIQ	3
6	134	138	GLHAP	5
7	164	173	FSSEDNADAT	10
8	202	208	KEAAGVF	7
9	219	228	PFGPSNGKLC	10
10	233	242	VLSKDGTTWI	10
11	246	254	QVVGPPRKR	9
12	325	333	PVFQRNKGR	9
13	349	360	PSTLDTQTQLPE	12
14	378	389	CKISESPSYSPV	12

Table 3 Amino acid sequence of B cell epitope in the C-terminal halve of SpTrz2 protein

编号 No.	开始 Start	结束 End	肽段序列 Peptide sequence	氨基酸长度 Length/aa
1	9	14	SATCPT	6
2	48	53	GTNTEP	6
3	83	87	KANTN	5
4	128	130	TVT	3
5	167	177	YSGDTRPNEKL	11
6	193	198	FEDDLK	6
7	203	211	QRQHSTASE	9
8	232	248	RSYDADFLPPDWTIYPK	17
9	250	253	KTIY	4

Fig.4 Construction of recombinant plasmids of pET28a-sptrz2 and pET28a-sptrz2N M, DNA marker; 1, PCR product of sptrz2; 2, PCR product of sptrz2N; 3 and 4, Identification of recombinant plasmid pET28a-sptrz2 by PCR; 5 and 6, Identification of recombinant plasmid pET28a-spTrz2N by PCR.

Fig.5 SDS-PAGE analysis of induced expression of recombinant protein SpTrz2 and SpTrz2N M, Protein molecular weight marker; 1, SpTrz2 recombinant strain non-induced; 2, SpTrz2 recombinant strain induced with IPTG; 3, The supernatant of SpTrz2 recombinant strain induced with IPTG; 4, The pellet of SpTrz2 recombinant strain induced with IPTG, the arrow indicated recombinant protein SpTrz2; 5, SpTrz2N recombinant strain non-induced; 6, SpTrz2N recombinant strain induced with IPTG; 7, The supernatant of SpTrz2N recombinant strain induced with IPTG; 8, The pellet of SpTrz2N recombinant strain induced with IPTG, the arrow indicated recombinant protein SpTrz2N.

Fig.6 Western blotting of polyclonal antibodies of SpTrz2 and SpTrz2N protein T, Total cell extracts; PMS, Postmitochondrial supernatants; Mt, Mitochondria.

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Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe

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