J Bacteriol Virol.  2010 Sep;40(3):123-130. 10.4167/jbv.2010.40.3.123.

Mouse Dual Ig Domain Containing Cell Adhesion Molecule Protein Expression and Purification Using the Baculovirus Expression Vector System

• Park SW
• Choi JH
• Goo TW
• Kim SR
• Lee GG
• Kang SW

• KMID: 2055049
• DOI: http://doi.org/10.4167/jbv.2010.40.3.123

Abstract

A baculovirus expression vector system (BEVS) is used routinely to produce recombinant proteins in the milligram scale. Dual Ig domain containing cell adhesion molecule (DICAM) belongs to the type I class of transmembrane proteins. It consists of a signal peptide, two V-type Ig domains in the extracellular region, and a short cytoplasmic tail of 442 amino acids. To purify the recombinant DICAM protein from cells overexpressing the mouse full-length DICAM gene, recombinant baculovirus is infected and recovered in the Sf9 cells. As a result, mouse DICAM protein was efficiently expressed and extracted from the insect cells using the BEVS. This recombinant protein can be used in further studies for functional test of DICAM protein in the cells.

Keyword

Baculovirus expression vector system; Dual Ig domain containing cell adhesion molecule; Recombinant protein

MeSH Terms

Amino Acids
Animals
Baculoviridae
Cell Adhesion
Cytoplasm
Insects
Mice
Protein Sorting Signals
Proteins
Recombinant Proteins
Sf9 Cells
Amino Acids
Protein Sorting Signals
Proteins
Recombinant Proteins

Figure

• 

  Figure 1. Recombinant AcDF baculovirus with mouse DICAM gene. A. Recombinant baculovirus transfer plasmids contain a mouse DICAM gene under the control of an AcNPV polyhedrin (polh) promoter. DICAM gene cassette (6) inserted into the pBACgus4X-EGFP plasmid DNA with BamH I and Not I restriction enzymes is terminated by stop codon of vector and added 6× His amino acids to 3′-end region. pBACgus4X-EGFP vector contain the 0.7 kb Enhanced Green Fluorescence Protein (EGFP) downstream of the other polh promoter. B. Specifically, about 1.3 kb full length DICAM gene was cloned into the pBACgus4X-EGFP transfer vector. To confirm of our clone pBAC-DF plasmid, vector was double digested by BamH I / Not I. Expected patterns were obtained, confirming targeted disruption. Lane 1, 1.0 kb DNA ladder; lane 2, pBACgus4X-EGFP; lane 3, pBAC-DF. C. Sf9 cells were infected with recombinant AcDF baculovirus and were examined and photographed by epifluorescence microscopy. Original magnification: × 100. D. RT-PCR analysis of DICAM expression with RNA extracted from different cells of AcDF infected and uninfected. A PCR product was detected for DICAM with RNA from AcDF infected Sf9 cells. Primers designed to amplify a 654 bp fragment of a Sf9 GAPDH were used for the internal control. Amplification for the GAPDH was detected following 35 cycles of PCR amplification. RNA extracted from non-infected cells was used as a negative control, and no amplicons were detected after 35 cycles of PCR amplification. A 1.0 kb DNA ladder was run in Lane 1.

• 

  Figure 2. Western blot analysis of DICAM protein induction in Sf9 cells infected with the recombinant AcDF baculovirus. A. For the selection of AcDF with DICAM, 6 different AcDF infected Sf9 cells were harvested at 6 days post infection and then examined for DICAM protein expression using anti-His-Ab (see Materials and Methods). Positions and molecular weights of protein size markers are indicated on the right (51 kDa; black bar). From Lane 1 to Lane 6 are represent #1 plaque and #6 plaque, respectively. Lane 7; Non-infected Sf9 cells. B. For the confirmation of recombinant DICAM protein purification condition, #1 selected recombinant AcDF from A was infected in Sf9 cells and harvested at 6 days post infection. Positions and molecular weights of protein size markers are indicated on the right (51 kDa; black bar). Lane 1; Culture media, Lane 2; Insoluble, Lane 3; Soluble, Lane 4; Beads after purification, and Lane 5; Mock.

Reference

1). Hitchman RB., Possee RD., King LA. Baculovirus expression systems for recombinant protein production in insect cells. Recent Pat Biotechnol. 2009. 3:46–54.

2). O'Reilly DR., Miller LK., Luckow VA. Baculovirus expression vectors: A laboratory manual. New York: Oxford University Press. 1994.

3). Philipps B., Forstner M., Mayr LM. A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells. Biotechnol Prog. 2005. 21:708–11.

4). Park SW., Lee HK., Kim TG., Yoon SK., Paik SY. Hepatocyte-specific gene expression by baculovirus pseudotyped with vesicular stomatitis virus envelope glycoprotein. Biochem Biophys Res Commun. 2001. 289:444–50.

5). Aucoin MG., Jacob D., Chahal PS., Meghrous J., Bernier A., Kamen AA. Virus-like particle and viral vector production using the baculovirus expression vector system/insect cell system: adeno-associated virus-based products. Methods Mol Biol. 2007. 388:281–96.

6). Bishop DHL., Entwistle PF., Cameron IR., Allen CJ., Posse RD. Field trials of genetically engineered baculovirus pesticides. Sussman M, Collins FA, Skinner FA, Stewart-Tull DE, editors. editors.The Release of Genetically Engineered Microorganisms. New York: Academic Press;1998. p. 143–79.

7). Jung YK., Jin JS., Jeong JH., Kim HN., Park NR., Choi JY. DICAM, a novel dual immunoglobulin domain containing cell adhesion molecule interacts with alphavbeta3 integrin. J Cell Physiol. 2008. 216:603–14.

8). Fraser MJ. The baculovirus-infected insect cell as a eukaryotic gene expression system. Curr Top Microbiol Immunol. 1992. 158:131–72.

9). Lebacq-Verheyden AM., Kasprzyk PG., Raum MG., Van Wyke Coelingh K., Lebacq JA., Battey JF. Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor. Mol Cell Biol. 1988. 8:3129–35.

10). Hollister J., Conradt H., Jarvis DL. Evidence for a sialic acid salvaging pathway in lepidopteran insect cells. Glycobiology. 2003. 13:487–95.

11). Jarvis DL. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology. 2003. 310:1–7.

12). Weyer U., Possee RD. A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells. J Gen Virol. 1991. 72:2967–74.

13). Kim JY., Choi JW., Park SW., Lee SG., Kim JM., Jheong WH, et al. Purification of protein expressed from three different regions of Norovirus (NoV). J Bacteriol Virol. 2008. 38:235–7.

14). Chang GD., Chen CJ., Lin CY., Chen HC., Chen H. Improvement of glycosylation in insect cells with mammalian glycosyltransferases. J Biotechnol. 2003. 102:61–71.

15). Morais VA., Costa MT., Costa J. N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells. Biochim Biophys Acta. 2003. 1619:133–8.

16). Palomares LA., Joosten CE., Hughes PR., Granados RR., Shuler ML. Novel insect cell line capable of complex N-glycosylation and sialylation of recombinant proteins. Biotechnol Prog. 2003. 19:185–92.

17). Yun EY., Goo TW., Kim SW., Choi KH., Hwang JS., Kang SW, et al. Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-mediated gene expression in insect cells. Biotechnol Lett. 2005. 27:1035–9.