Expression of partially N-linked glycoproteins using the 1-Step Human Coupled in vitro Translation System
by Krishna Vattem, Ph.D. - 03/24/14
Glycosylation is a form of co-translational and post-translational modification in which sugars are added onto proteins. Glycosylation is important for protein function, protein folding and cell-cell adhesions. The Thermo Scientific 1-Step Human Coupled IVT Kit (Part No. 88881) contains HeLa cell lysate and other components to supply the cellular machinery required to support protein translation from DNA templates. In this brief communication, I provide a protocol and demonstrate that the IVT reaction mixture of this system contains not only just translational machinery but also the necessary sugars and functional elements to glycosylate the proteins being expressed. I examined expression of three different glycoproteins and also used endoglycosidase H, an enzyme that cleaves asparagine-linked mannose rich oligosaccharides, to determine the type of glycosylation that is occurring in human IVT system [1-3].
Protocol for glycoprotein expression
Take precautions to maintain an RNAse-free environment. Thaw reagents immediately before use and keep on ice. Assemble the IVT reaction at room temperature. Promptly store any unused HeLa Lysate and other kit components at -80°C. (This protocol differs from the default protocol in product instructions in the DNA concentration used and the time and temperature of incubation.)
- Thaw on ice the HeLa Lysate, Accessory Proteins, Reaction Mix [components of the 1-Step Human IVT Kit (Part No. 88881)] and glycoprotein encoding DNA diluted to a concentration of 0.1µg/µL. To hasten the thawing process, warm vials in gloved hands. Once thawed half way, place back on ice.
- Prepare reaction at room temperature in the proportions specified in Table 1. Add the reagents in the order listed into a 1.5mL nuclease-free tube. Gently mix the reaction after each reagent addition. If scaling up the reaction, maintain the specified proportions.
Table 1. Components for the IVT reaction (for glycoprotein expression). Named components are from the Thermo Scientific 1-Step Human Coupled IVT Kit (Part No. 88881).
- Incubate the reaction for 3 to 4 hours at 26°C.
- Following the translation reaction, maintain reactions on ice for same-day downstream applications and analysis. For long-term storage, maintain reactions at ≤ -20°C. Completed reactions may be centrifuged at 10,000 × g for 5 minutes to gather contents.
Three different glycoproteins human chorionic gonadotropin hormone-beta subunit (HCG-β), alpha-1-acid glycoprotein 1 (ORM1), and erythropoietin (EPO) cloned into pT7CFE-CHA vector (Part No.88862) were expressed in the 1-Step Coupled Human IVT system. Lysates expressing these glycoproteins were then treated with endoglycosidase H (Endo H) according to the manufacturer’s instructions. Treatment with Endo H eliminated the higher MW band in SDS-PAGE, suggesting that the proteins expressed in human IVT are glycosylated (Figure 1).
Endo H only cleaves non-complex N-linked saccharides; therefore, these results indicate that only N-linked glycosylation occurs in the human IVT system. We did not observe O-linked glycosylation of fetuin, a glycoprotein containing both N-linked and O-linked glycans, when expressed in human IVT system and enzymatically digested with O-glycosydases (data not shown). Further analysis is underway to characterize the type of N-linked glycosylation using mass spectrometry methods.
Figure 1. Expression of N-linked glycoprotein using Human IVT. DNAs (0.1 or 0.2μg) of HA-tagged HCGβ, ORM1 or EPO were added to 25μL coupled human IVT reactions and incubated at 26°C for 4 hours. The reactions were left untreated or treated with Endo H according to the manufacturer’s instructions and proteins separated on SDS-PAGE gel and probed for HA tag using anti-HA antibodies and western blot. The position of glycosylated (triangle markers) and unglycosylated (lowest bands) proteins are indicated. Endo H completely removes all the N-linked mannose residues resulting in the appearance of proteins as a single band. The relative proportion of glycosylated vs. unglycosylated proteins was significantly higher with lower DNA concentrations.
I have demonstrated that partially N-linked glycoproteins can be expressed in the Thermo Scientific 1-Step Human Coupled IVT System. Glycoprotein expression in the system requires optimizing the reaction conditions from the default protocol provided in the product instructions. Expression of glycosylated protein was maximized by reducing the burden on protein expression by (1) decreasing the amount of DNA added to the reaction, (2) decreasing the incubation temperature to 26°C and (3) reducing the incubation time to less than 4 hours. This technique provides researchers with an in vitro method to induce glycosylation as a post-translational modification of proteins.
Protein expression was performed using the protocol specified above. Determination of the type of glycoprotein linkage in the proteins expressed was carried out by treating the samples with Endo H (#P0702S, New England Biolabs) according to the manufacturer’s instructions. Briefly, 5µL of IVT reaction expressing glycoprotein was combined with 1μL of 10X Glycoprotein Denaturing Buffer and the volume was brought up to 10μL with deionized water. Glycoproteins were then denatured by heating the reaction at 95 to 100°C for 10 minutes. The contents were spun down briefly at 500Xg for 30 seconds. The total volume of this reaction volume was brought up to 20μL by adding 2μL of 10X G5 Reaction Buffer, 3μL Endo H and water. Reactions were incubated at 37°C for 1 hour. Finally, proteins were separated by SDS-PAGE, blotted to membrane, probed for the HA-tag of the proteins using anti-HA antibody (Ab No. 26183), and detected using a Fast Western Blot Kit (Part No. 35065).
- Robbins, P.W., et al. (1984). Primary structure of the streptomyces enzyme endo-beta-N-acetylglucosaminidase H. J. Biol. Chem. 259: 7577-83.
- Trumbly, R.J., et al. (1985). Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product. J. Biol. Chem. 260: 5683-90.
- Mikami, S., et al. (2006). A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins. J. Biotechnol. 127: 65-78.
Thermo Scientific 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100 µg/mL per reaction.
The Human IVT Kits are unique HeLa cell lysate-based protein expression systems for in vitro translation or coupled transcription/translation reactions. Protein expression is performed in a single 90-minute reaction that can be extended for up to 6 hours with continued protein production up to 100 µg/mL when combined with the optimized pT7CFE1 Expression Vector. The Human IVT Kits can express functional enzymes, phosphoproteins, glycoproteins and membrane proteins for immediate use in studying protein interactions, performing rapid mutational analysis and measuring activity.
Features of the 1-Step Human Coupled IVT Kit:
- Functional—uses the human translational machinery to express active proteins
- Convenient—perform transcription and translation in a single step
- High performance—greater yields compared to rabbit reticulocyte in vitro translation
- Reliable—express proteins that fail in rabbit reticulocyte systems
For Research Use Only. Not for use in diagnostic procedures.