Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris†

doi:10.7503/cjcu20131071

Abstract

Abstract:

Ganoderma lucidum immunoregulatory protein(LZ-8) was recombinant expressed in Pichia pastoris by prolonging its in vivo half-life through genetic fusion with human IgG-Fc fragment. High purify of LZ8-Fc was obtained by affinity chromatography and size-exclusion chromatography. The result of hemagglutination reaction indicated that LZ-8’s immunoregulatory activity was inhibited by IgG Fc fragment. Molecular dyna-mics simulation combined with SEC-HPLC and CD proved that LZ8-Fc assembled itself in the same way with LZ-8, dimer in natural state. All of the results suggested that LZ-8 in N-terminal affected IgG-Fc fragment in downstream, which is supposed an important issue in the recombinant expressing of fusion protein. The results will provide a solid basis for preparing and optimizing technology of fusion protein.

Key words: LZ8-Fc, Molecular dynamics, Dimer

CLC Number:

O629
O641

TrendMD:

HOU Yue, SUN Fei, JIN Jing, LIU Zhiyi, ZHENG Qingchuan, LIANG Chongyang. Expression and Molecular Dynamics of Recombinant Ganoderma Lucidum Immunoregulatory Protein-IgG Fc(LZ8-Fc) Fusion Protein in Pichia pastoris^†[J]. Chem. J. Chinese Universities, 2014, 35(2): 303.

Figures/Tables 7

Fig.1 Construction and identification of pPICZαA-LZ8-Fc(A) Schematic of the pPICZαA-LZ8-Fc. LZ8-Fc sequence was cloned into pPICZαA via two restriction enzyme sites, EcoR I and Xho I, which next to α-factor secretion signal.(B) Identification of the pPICZαA-LZ8-Fc by PCR. Lane M: DNA marker; lanes 1 and 2: samples from the same PCR conditions with different tubes. The arrow points out the location of LZ8-Fc sequence.

Fig.2 SDS-PAGE(A), Western blotting(B) and ESI-TOF/TOF analysis(C) of LZ8-Fc expressed in P. pastoris strains X33(A) Lane M: protein molecular weight marker; lane 1: serum albumin; lane 2: LZ-8 protein; lanes 3—9: samples taken from the culture supernatant induced for 48, 42, 36, 30, 24, 12 and 6 h, respectively, with 0.5% methanol and separated by 0.12 mg/mL SDS-PAGE gel. (B) Lanes 1—7 were the same sequences of those in lanes 3—9 in (A). (C) Red fold words mean high abundance.

Fig.3 Two-steps chromatography process(A,B), SDS-PAGE(C) and IEF analysis(D) for LZ8-Fc(A, B) The arrows point to the peak of LZ8-Fc; (C) the single band(arrow) shows the same position with LZ8-Fc; (D) the band of LZ8-Fc(the arrow) shows the same position with the marker(pI=7.0).

Fig.4 Hemagglutination assay of LZ8-Fc and LZ-8LZ8-Fc and LZ-8 were diluted from 50 μg/mL to 1 μg/mL(row 1 to 8), columns a and b: LZ8-Fc; columns c and d: LZ-8.

Fig.5 SEC-HPLC analysis of LZ8-Fc’s assembling model(A) Purity analysis of the LZ8-Fc by HPLC. (B) Peaks 1—5 showed the absorption of the standard proteins, the molecular weights are 670, 158, 44, 17 and 1.35, respectively.

Fig.6 Western blotting(A), structure model(B) and molecular dynamics analysis result of bond length(2000 ps) for LZ8-Fc(A) Lane 1: LZ8-Fc concentration of 0.3 mg/mL; lane 2: concentrated LZ8-Fc was 2.5 mg/mL.(B) Structure model of LZ8-Fc, which connected with GGSS. (C, D) Distance of two peptides, which in (B) marked C and D.

Fig.7 CD spectrum(A) and analysis of secondary structure(B) of LZ8-Fc protein

References 21

[1]	Zhu R. Y., Xin X., Dai H. Y., Li Q., Lei J. Y., Chen Y., Jin J., Protein Expres. Purif., 2012, 85, 32—37
[2]	Wang D. D., Su M. M., Sun Y., Huang S. L., Wang J., Yan W. Q., Protein Expres. Purif., 2012, 86, 75—81
[3]	Zhang X. P., Sun F., Liu Z. Y., Zhang S. Q., Liang C. Y., Mol. Med. Rep., 2013, 7(3), 975—980
[4]	Karri V., Pulugurtha B. K., Funct. & Integr. Genomics, 2013, 13(4), 435—443
[5]	Meng X. Y., Zheng Q. C., Zhang H. X., Proteins and Proteomics, 2009, 1794(7), 1066—1072
[6]	Shi Z., Wang Z. J., Xu H. L., Tian Y., Li X., Bao J. K., Sun S. R., Yue B. S., Comput. Biol. Chem., 2013, 47(12), 56—65
[7]	Sangeeta K., Debjani R., J. Mol. Graph. Model., 2009, 27(8), 871—880
[8]	Tanaka S., Ko K., Kino K., Tsuchiya K., Yamashita A., Murasugi A., Sakuma S., Tsunoo H., J. Biol. Chem., 1989, 264(1), 472—478
[9]	van der Hem L. G., van der Vliet J. A., Kino K., Hoitsma A. J., Tax W. J., Transplant Proc., 1996, 28, 958—959
[10]	Hsu H. C., Hsu C. I., Lin R. H., Kao C. L., Lin J. Y., Biochem. J., 1997, 323(Part 2), 557—565
[11]	Wang P. H., Yang S. F., Chen G. D., Han C. P., Chen S. C., Lin L. Y., Ko J. L., Reprod. Sci., 2007, 14(5), 475—485
[12]	Liao C. H., Hsiao Y. M., Sheu G. T., Chang J. T., Wang P. H., Wu M. F., Shieh G. J., Hsu C. P., Ko J. L., Biochemical Pharmacology, 2007, 74, 1541—1554
[13]	Jinn T. R., Wu C. M., Tu W. C., Ko J. L., Tzen J. T., Biosci. Biotechnol. Biochem., 2006, 70(11), 2627—2634
[14]	Lin Y. L., Liang Y. C., Tseng Y. S., Huang H. Y., Chou S. Y., Hseu R. S., Huang C. T., Chiang B. L., Journal of Leukocyte Biology, 2009, 86(10), 877—889
[15]	Chu C. L., Chen D. C., Lin C. C., Human Vaccines, 2011, 7(11), 1161—1164
[16]	Huang L., Sun F., Liang C. Y., He Y. X., Bao R., Liu L. X., Zhou C. Z., Proteins, 2009, 75(2), 524—527
[17]	Lin W. H., Hung C., Hsu C. I., Lin J. Y., J. Biol. Chem., 1997, 272(32), 20044—20048
[18]	Berend T., Lisa S., Richard J. B., Kerry A. C., Nature Protocols., 2006, 1(2), 1006—1021
[19]	Frederick M.A., Roger B., Robert E. K., David D. M., Translated by Chen B. M., Ma X. J., Shu Y. L., Short Protocols in Mole-cular Biology, Science Press, Beijing, 2005, 378—407
	(马学军, 舒跃龙[译]. 精编分子生物学实验指南, 北京: 科学出版社, 2005, 378—407)
[20]	Chu W. T., Zhang J. L., Zheng Q. C., Chen L., Zhang H. X., PLoS One, 2013, 8(5), 1—9
[21]	Yu J., Zhou C. J., Proteins, 2007, 68(4), 972—979