Prokaryotic expression

Molecular cloning and characterization, and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken

T. Zhang, Zhang, G. X., Han, K. P., Tang, Y., Wang, J. Y., Fan, Q. C., Chen, X. S., Wei, Y., and Wang, Y. J., Molecular cloning and characterization, and prokaryotic expression of the GnRH1 gene obtained from Jinghai yellow chicken, vol. 14, pp. 2831-2849, 2015.

The gonadotropin-releasing hormone (GnRH) plays an important role in the control of reproductive functions. Recent studies have reported the occurrence of GnRH molecular variants in numerous species. In this study, the GnRH1 gene from Jinghai yellow chicken was cloned by reverse transcriptase-polymerase chain reaction and transformed into BL21 (DE3) competent cells. The GnRH1 gene and amino acid sequences were subjected to bioinformatic analyses.

Study of Hgp44 from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6 and IL-8

J. Zhang, Cui, H. H., Wang, Y., Zhang, Q. X., Deng, S. L., Chen, Y. D., and Chen, H., Study of Hgp44 from Porphyromonas gingivalis on inducing HUVECs to secrete IL-6 and IL-8, vol. 13, pp. 2208-2219, 2014.

The aim of this study was to clone, express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis and purify the protein. Furthermore, the effect of Hgp44 from P. gingivalis on inducing HUVECs to secrete IL-6 and IL-8 was evaluated. The Hgp44 gene fragment was amplified by polymerase chain reaction, and then inserted into the cloning vector pMD18-T and linked with a prokaryotic expression vector pET22b to construct the recombinant expression plasmid pET22b-Hgp44.

Expression of the porcine lipoic acid synthase (LIAS) gene in Escherichia coli

W. F. Lu, Cao, J. J., Guo, Y. J., Zhong, K., Zha, G. M., Wang, L. F., and Yang, G. Y., Expression of the porcine lipoic acid synthase (LIAS) gene in Escherichia coli, vol. 13, pp. 5369-5377, 2014.

Lipoic acid synthase, which exists primarily in mitochondria, participates in the biosynthesis of intrinsic lipoic acid. The lipoic acid synthase gene in pig is known as LIAS. To further investigate the biological functions of the protein that is encoded by LIAS, we cloned the open read frame of porcine LIAS (GenBank No. JN797612.1) into the expression vector pET-28α(+). The resulting pET-28α(+)-Lias recombinant vector was introduced into the Escherichia coli BL21 (DE3) strain.

Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purification, and preparation of a polyclonal antibody

H. L. Zheng, Li, H., Sun, Y. S., Yang, Z. Y., and Yu, Q., Parathyroid hormone-related peptide (PTHrP): prokaryotic expression, purification, and preparation of a polyclonal antibody, vol. 13, pp. 6448-6454, 2014.

Parathyroid hormone-related peptide (PTHrP) plays important roles in promoting cancer occurrence and in the development of bone metastases. To increase our knowledge of the biological functions of PTHrP, the prokaryotic expression vector pET-PTHrP was successfully constructed and the His-PTHrP fusion protein was expressed in Escherichia coli. Anti-PTHrP polyclonal antibody was then prepared from rabbits. Finally, the goat tissue expression profile of PTHrP was analyzed by Western blot with the anti-PTHrP polyclonal antibody.

Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear

J. Tang, Lin, J., Zhang, B. L., Wang, Z. H., Li, X. G., and Chang, Y. H., Cloning and expression analysis of PpSUT2 encoding a sucrose transporter in pear, vol. 13, pp. 8932-8945, 2014.

A 1794-bp cDNA fragment was amplified from mRNA isolated from pear (Pyrus pyrifolia NaKai. Cuiguan) leaves by using primers based on the sequences generated during the analysis of the pear transcriptome. The 597-amino acid sequence encoded by the cDNA was compared with the sequences in GenBank, and it was found to be similar to that of members of the sucrose-proton co-transporter family. The hydrophobic protein, which was predicted to have 11 transmembrane domains, was designated as PpSUT2.

Cloning and prokaryotic expression of rat homolog of Serpina3n and its expression change during liver regeneration

G. P. Wang, Zhang, X. S., Li, Y. H., Zheng, J. L., Tang, C. Z., and Zhang, W. X., Cloning and prokaryotic expression of rat homolog of Serpina3n and its expression change during liver regeneration, vol. 11, pp. 3175-3185, 2012.

A strikingly upregulated expressed sequence tag was screened from regenerating rat liver at 8 h in a 0-4-8-12 h short-interval successive partial hepatectomy model from a previous study. In the present study, a full-length open reading frame (ORF) corresponding to this expressed sequence tag was predicted through electronic cloning and was subsequently cloned from an 8-h rat regenerating liver and deposited in GenBank (accession No. HM448398). Sequence analysis of HM448398 and the predicted ORF revealed that the two ORFs may be different transcripts of a gene.

Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein

Y. Yi, Nian, Y. - Y., Ji, H. - W., Zhang, H., Zhu, L., and Xu, Z. - W., Coexpression of interleukin-6 and -2 from giant panda in Escherichia coli and the biological activity of the fusion protein, vol. 12, pp. 1987-1995, 2013.

To construct a fusion cytokine protein with more and stronger bioactivities to enhance the immunity of the cytokine alone, we expressed interleukin (IL)-6/(IL)-2 from giant panda (Ailuropoda melanoleuca) in Escherichia coli as a 59.4-kDa fusion protein. Subsequently, the inclusion bodies were solubilized with 8 M urea and applied onto a Ni-nitrilotriacetic acid column. The final production of IL-6/IL-2 reached 6 mg/L in soluble form, and the purified final product was >96% pure.

Molecular cloning and expression of the porcine S14R gene in Escherichia coli

Y. J. Guo, Liu, G. Z., Wang, C. M., Wang, Y. Y., Li, H. J., Zhong, K., Lu, W. F., Wang, Y. L., and Yang, G. Y., Molecular cloning and expression of the porcine S14R gene in Escherichia coli, vol. 12, pp. 4405-4412, 2013.

We amplified S14R protein gene cDNA of porcine, cloned it into a prokaryotic expression plasmid, and expressed it in Escherichia coli. A pair of primers was designed based on the cDNA sequence of the porcine S14R gene in GenBank. The target gene fragment from porcine liver tissue was amplified by RT-PCR. Confirmed by auto-sequencing, the target gene fragment was subcloned into an expression vector of pET28a. The pET28a-S14R construct was subsequently transformed into E. coli BL21 (DE3).

Expression and purification of GST-FHL2 fusion protein

H. Yu, Ma, Q., Lin, J., Sun, Y. F., and Zheng, F., Expression and purification of GST-FHL2 fusion protein, vol. 12, pp. 6372-6378, 2013.

Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis.

Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA

J. B. Cajazeiras, Melo, L. M., Albuquerque, E. S., Rádis-Baptista, G., Cavada, B. S., and Freitas, V. J. F., Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA, vol. 8, pp. 1147-1157, 2009.

Low purification efficiency and incomplete characteriza­tion of male goat (buck) spermadhesins (Bdhs) prompted us to develop an effective system to produce recombinant Bdhs (rBdhs). Bdh-2 cDNA was inserted into a prokaryotic expression plasmid, pTrcHis TOPO. The pTrcHis-Bdh-2 system was constructed to produce a His6 fusion protein in Escherichia coli Top10 cells. Recombinant clones were se­lected by growth in ampicillin-enriched medium, PCR amplification and nucleotide sequencing.

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