Production of human type II collagen using an efficient baculovirus-silkworm multigene expression system.
Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257 bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure. About 1 mg/larva human type II collagen is purified from silkworm skin, which shows a putative large scale of collagen production way. An activity assay of recombinant human type II collagen expressed by silkworm larvae demonstrated that the recombinant protein has considerable bioactive properties. Scanning electron microscopy of purified proteins clearly reveals randomly distributed and pitted structures. We conclude that the baculovirus-silkworm multigene expression system can be used as an efficient platform for express active human type II collagen and other complicated eukaryotic proteins.
Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China.
Qi
Q
Qi
Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China.|Henan Key Laboratory of Ecological Security for Water Source Region of Mid-line Project of South-to-North Diversion, Nan Yang Normal University, Nanyang, 473000, Henan, China.
Lunguang
L
Yao
Institute of Biomedicine, Jinan University, Guangzhou, 510632, Guangdong, China.
Zhisheng
Z
Liang
Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China.
Donghua
D
Yan
Medical Technology College of Nanyang Medical University, Nanyang, 473041, Henan, China.
Zhuo
Z
Li
Institute of Biomedicine, Jinan University, Guangzhou, 510632, Guangdong, China.
Yadong
Y
Huang
Subtropical Sericulture and Mulberry Resources Protection and Safety Engineering Research Center, Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, 510642, Guangdong, China. cyfz@scau.edu.cn.
Jingchen
J
Sun
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D012831:Bombyx / Q000235:genetics / Q000378:metabolism / Q000821:virology*; D024043:Collagen Type II / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors; D006801:Humans; D008855:Microscopy, Electron, Scanning; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D061987:Sf9 Cells
D024043:Collagen Type II; D011994:Recombinant Proteins
BEVS; Bombyx mori; Human type II collagen; Sf9; Triple-helix
Microsc Res Tech. 1994 Aug 1;28(5):378-84
7919525
Appl Biochem Biotechnol. 1993 Aug-Sep;42(2-3):137-59
8250545
Matrix Biol. 1998 Jan;16(6):329-38
9503366
Acta Ophthalmol. 2009 Mar;87(2):193-8
18803623
EMBO J. 1997 Nov 17;16(22):6702-12
9362485
Matrix Biol. 1999 Feb;18(1):89-103
10367734
J Biotechnol. 2010 Jan 1;145(1):23-9
19835917
PLoS One. 2012;7(2):e31199
22319617
Appl Microbiol Biotechnol. 2003 Jul;62(1):1-20
12733003
Hum Cell. 1988 Jun;1(2):150-61
2979194
Biotechnol Appl Biochem. 2010 Dec;57(3):117-25
20979580
Diabetologia. 1996 Jul;39(7):800-6
8817104
Biotechnol Prog. 2009 Mar-Apr;25(2):524-9
19301249
Arthritis Res Ther. 2004;6(3):R213-9
15142267
Matrix Biol. 2014 Feb;34:105-13
24113490
J Am Acad Dermatol. 2006 Feb;54(2):258-65
16443056
Dev Biol Stand. 1999;99:25-34
10404872
J Biomed Mater Res B Appl Biomater. 2004 Nov 15;71(2):343-54
15386396
Semin Arthritis Rheum. 1991 Dec;21(3 Suppl 2):2-11
1796302
PLoS One. 2012;7(3):e32510
22403668
Glycoconj J. 1999 Feb;16(2):109-23
10612411
Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3028-33
16488977
Ann Plast Surg. 1984 Jul;13(1):9-14
6331269