Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Sayan Chakraborty; Krittin Trihemasava; Guozhou Xu

doi:10.3791/58283

Pflanzliche Proteine in Insektenzellen abgesondert ändern Baculovirus Expressionsvektoren herstellen

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Biochemistry

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JoVE Journal Biochemistry

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

Pflanzliche Proteine in Insektenzellen abgesondert ändern Baculovirus Expressionsvektoren herstellen

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09:14 min

August 20, 2018

DOI: 10.3791/58283-v

Sayan Chakraborty¹, Krittin Trihemasava¹, Guozhou Xu¹

¹Department of Molecular and Structural Biochemistry, College of Agriculture and Life Sciences,North Carolina State University

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Overview

This article presents protocols for using the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for crystallization. The method is advantageous for producing recombinant proteins at a low cost, facilitating structural biology studies.

Key Study Components

Area of Science

Biochemistry
Structural Biology
Protein Crystallization

Background

Utilization of insect cells for protein expression.
Modification of baculovirus expression vectors.
Importance of secreted proteins in structural studies.
Cost-effectiveness of the method for large-scale protein production.

Purpose of Study

To provide protocols for producing plant secreted proteins.
To facilitate protein structure determination through crystallography and cryo-EM.
To enhance understanding of secreted protein functions.

Methods Used

Synthesis of DNA fragments with specific cutting sites.
Digestion of DNA with restriction enzymes.
Ligation of DNA fragments to expression vectors.
Incubation and transformation into insect cells.

Main Results

Successful production of recombinant secreted proteins.
Demonstration of low-cost protein expression.
Potential for high yields suitable for structural analysis.
Protocols validated for reproducibility in research settings.

Conclusions

The insect cell and baculovirus system is effective for protein production.
This method supports advancements in structural biology.
Future applications may enhance understanding of protein functions.

Frequently Asked Questions

What is the main advantage of this protein expression system?

The main advantage is the ability to produce large amounts of recombinant secreted proteins at a relatively low cost.

How does this method contribute to structural biology?

It facilitates the determination of protein structures through techniques like crystallography and cryo-EM.

What are the key steps in the protocol?

Key steps include synthesizing DNA fragments, digesting with restriction enzymes, and ligating to expression vectors.

Can this method be used for any type of protein?

It is specifically designed for plant secreted proteins, but the principles may apply to other proteins as well.

What is the role of the baculovirus in this system?

The baculovirus acts as a vector to facilitate the expression of the target proteins in insect cells.

Is this method widely used in research?

Yes, it is a common method in biochemistry and structural biology for producing proteins for analysis.

Hier präsentieren wir Ihnen die Protokolle zur Nutzung von Insekten Zell- und Baculovirus Protein Expressionssystems zu produzieren große Mengen an Pflanzeneiweiß abgesondert für Protein Kristallisation. Ein Baculovirus Expressionsvektor wurde entweder mit GP67 oder Insekt Hemolin Signalpeptid für Pflanze Sekretion Proteinexpression in Insektenzellen geändert.

Diese Methode kann dazu beitragen, Schlüsselfragen im Bereich der Biochemie und Strukturbiologie sezernierter Proteine zu beantworten, wie z. B. die Bestimmung der Proteinstruktur durch Kristallographie und Kryo-EM. Der Hauptvorteil dieser Technik besteht darin, dass sie eine große Menge an rekombinanten sekretierten Proteinen mit relativ geringen Kosten für die Bestimmung der Proteinstruktur produziert. Zuerst wird ein DNA-Fragment synthetisiert, das eine BglII-Schneidstelle mit fünf Primzahlen enthält, die Sekretionssignalsequenz von Interesse an einer Multiklonierungsstelle, wie im Textprotokoll beschrieben.

Als nächstes verdauen Sie vier Mikrogramm der DNA mit BglII und X1 und vier Mikrogramm einer Expressionsvektor-DNA mit BamH1 und X1. Mischen Sie 10 Einheiten jedes Restriktionsenzyms mit der DNA in einem Reaktionspuffer mit 1X-Konzentration. Inkubieren Sie vier Stunden lang bei 37 Grad Celsius. Geben Sie 10 Mikroliter eines Ligationsreaktionsgemisches, das 1X T4 DNA Ligase-Reaktionspuffer und fünf Einheiten T4 DNA Ligase enthält, in ein frisches 1,5-Milliliter-Eppendorf-Röhrchen.

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Biochemie Ausgabe 138 Insekt-Zelle Baculovirus Expressionsvektor sekretorischen Protein signal-Peptid GP67 Hemolin Bacmid Transfektion Zellkultur Glykosylierung Proteinexpression Protein Kristallisation