Insect Cell Biotechnology

Gordon D. O. Maramorosch

• Publisher: CRC Press
• Year: 2018
• ISBN:
  9781351090476
• Edition: 1
• Topic: Biological Sciences
• Subtopic: Entomology
• Index: Biological Sciences

Insect Cell Biotechnology provides a lucid, up-to-date description of recent major advances in the field. A number of significant topics are addressed, including the use and production of baculoviruses in insect cells, baculovirus specificity, bacterial toxin studies in cultured insect cells, scale-up operations required in the production of recombinant protein and insect viruses propagated in insect cells, growth and nervous system interactions, and the physiological and developmental capacities of cell lines. Transfection in Drosophila cells and a chapter on the theoretical and practical implications of stress produced by x-rays, ultraviolet light, chemicals, psoralens, and heat are discussed as well.

Chapter 1

EXPRESSION OF FOREIGN GENES IN INSECT CELLS USING BACULOVIRUS VECTORS

Susumu Maeda

TABLE OF CONTENTS

I. Introduction

II. Baculovirus Expression Vectors

A. Organization and Expression of the Baculovirus Genome

B. Baculovirus Expression Vector Systems

1. Advantages

2. Construction of Recombinant Baculoviruses

3. Foreign Gene Constructs for Insertion into Baculovirus Transfer Vectors

C. Promoters Used to Drive Foreign Gene Expression

1. Polyhedrin Promoter

2. p10 Promoter

3. Other Baculovirus Late Gene Promoters

4. Baculovirus Early Gene Promoters

5. Chimeric and Modified Promoters

6. Host Insect and Nonbaculovirus Promoters

D. Expression Vectors Generating Polyhedron-Deficient Viruses

1. Traditional Transfer Vectors

2. Polyhedrin Fusion Transfer Vectors

3. Transfer Vectors for Coexpression of Two Genes

E. Improved Strategies for the Isolation and Identification of Recombinant Viruses

1. Isolation by Dilution Methods and DNA Hybridization

2. Linearized Parental Viral DNA

3. β-Galactosidase Marker

4. Polyhedron-Positive Marker

5. Baculovirus-Yeast Shuttle Vectors

6. Other Isolation Strategies

F. Host-Dependent Foreign Gene Expression

III. Characteristics of Expressed Foreign Gene Products

A. Proteolytic Processing

1. Signal Sequence Cleavage and Secretion

2. Internal Proteolytic Cleavage

B. Glycosylation

C. Phosphorylation

D. C-Terminus α-Amidation

E. Fatty Acid Acylation

F. Solubility

G. Oligomerization

H. Assembly of Viral Particles

IV. Expression of Foreign Genes in Larval Insects

V. Foreign Gene Expression for Pest Insect Control

VI. Concluding Remarks

Acknowledgments

References

Susumu Maeda, Ph.D., is Associate Professor of Entomology at the University of California, Davis. He received his B.S. in Entomology/Agrobiology, in 1975 and Ph.D. at the University of Tokyo, in 1983. Postdoctoral fellow at U.C. Berkeley, 1980-1981; Assistant Professor at Tottori University, Japan, 1978-1988; Visiting Research Scientist, Zoecon Research Institute, Sandoz Crop Protection in Palo Alto, California, 1987-1988; Assistant Professor at U.C. Davis, 1988-1991; Associate Professor at U.C. Davis, 1991-present. American Society of Microbiology, Society of Invertebrate Pathology, American Society of Virology, American Entomological Society, American Society of Biochemistry and Molecular Biology. Molecular biology of insect viruses and their molecular interactions to host insects; application of recombinant insect viruses in medical and agricultural sciences.

I. Introduction

Baculoviruses (family Baculoviridae) are characterized by circular double-stranded DNA genomes that are encapsulated within rod-shaped enveloped virions. They have been isolated exclusively from insects and crustaceans. Baculoviruses are classified into three genera: nuclear polyhedrosis virus (NPV), granulosis virus, and non-occluded baculovirus. During an early stage of infection, NPVs produce viral particles (budded virions) that are involved in cell-to-cell infection (secondary infection) in both larval tissues and established cell lines. During a late stage of infection, NPVs have a unique feature of producing inclusion bodies called polyhedra in the infected nucleus. Polyhedra protect the numerous progeny viral particles (occluded virions) embedded within them and are required for oral (per os) infection. Upon ingestion, polyhedra are broken down by alkaline proteinases in the insect gut juices and release occluded virions, which subsequently infect midgut tissues by fusion. The major component of polyhedra is a very highly expressed (roughly 30% of total proteins at a late stage of infection) protein called polyhedrin. The polyhedrin gene is nonessential for viral replication. Using the polyhedrin promoter, baculoviruses (NPVs) have been recognized as efficient vectors for the high-level expression of foreign genes in insect cell lines and larvae. Autographa californica NPV (AcNPV) followed by Bombyx mori NPV (BmNPV) is the most commonly used baculovirus vector for foreign gene expression experiments.

This chapter will emphasize recent (generally post-1989) progress in the use of baculovirus expression vectors, especially in regard to applications in agriculture and medicine, and outline the characteristics of baculovirus expressed proteins. The unique advantages of expression in insect larval systems will also be covered. Many general and in-depth reviews are available in the use¹, ², ³, ⁴, ^5,, ⁶, ⁷, ⁸, ⁹ and ¹¹ and methodology^12-16 of baculovirus expression vector systems for those wishing further background.

II. Baculovirus Expression Vectors

A. Organization and Expression of the Baculovirus Genome

The baculovirus genome is composed of 100 to 180 kbp of double-stranded circular DNA. About 50% of the nucleotide sequence of the AcNPV genome has been determined (see Reference 17). Several genes of the Orgyia pseudotsugata NPV genome have also been identified and sequenced (see Reference 18), and two laboratories have nearly completed sequencing the entire genomes of AcNPV¹⁹ and BmNPV.²⁰

Five discrete (hrl to hr5) homologous regions (hrs) are found in the NPV genome.^21-23 Hr4 of AcNPV²² and BmNPV,²³ and hr2 of BmNPV²³ can be subdivided into two smaller hrs. Each hr is 1 to 2 kbp long and possesses 3 to 7 repeats of a relatively conserved, 100 bp-long, AT-rich sequence with a 20 bp-long palindrome structure at its core.^22,23 In the case of AcNPV and BmNPV, a GAATTC sequence is found in the palindrome, which is recognized and cleaved by the restriction endonuclease EcoRI. Hrs function to enhance the expression of genes²² or act as replication origins for viral DNA replication.²⁴

Outside of the hrs, the NPV genome is generally covered by unique sequences consisting of coding regions and their 5′ and 3′ noncoding regulatory sequences (promoters, poly(A) signals, etc.). The noncoding regions generally range from a few to several hundred base pairs. Only one intron-possessing gene, IE-0, has been identified in the AcNPV genome.²⁵ The IE-0 gene product is detected only at an early stage of infection; however, it is unknown whether IE-0 is essential for viral replication. In most cases baculovirus open reading frames that correspond to expressed polypeptides do not overlap. A preferential gene direction and gene clusters with similar functions are not found (see Reference 14). Genes commonly found in viruses including those involved in virus structure (e.g., envelope (gp64) protein), DNA replication (e.g., DNA polymerase), and regulation of gene expression (e.g., IE-1) have been identified in the NPV genome (see Reference 14). Unique or unusual genes identified in the baculovirus genome include: UDP-glucosyltransferase,²⁶ apoptosis blocking protein,²⁷ chitinase,^28,29 and ubiquitin.³⁰

The expression of baculovirus genes is temporally regulated and characterized by three distinct phases: early, late, and very late. Early genes do not require the expression of other viral genes for basal levels of expression. Late and very late gene expression begin 6 to 8 and 20 to 24 hours postinfection, respectively, and require the expression of early (and/or late) gene products. Early genes are transcribed by the host RNA polymerase II complex, which is cc-amanitin sensitive.³¹ Late and very late genes are believed to be transcribed by a viral-coded or viral-induced (or modified) host RNA polymerase complex. The expression of most viral early and host genes declines with the onset of late gene expression. Host and viral (both early and late) gene expression is nearly completely terminated during the active expression of very late genes (36 to 72 hours postinfection). In AcNPV the most abundantly expressed very late gene is polyhedrin followed by the p10 gene. All baculovirus late gene promoters identified to date possess the consensus sequence (A/T/G)TAAG (see Reference 32). About half of all known baculovirus early gene promoters possess a CAGT motif 20 to 25 bp downstream of the TATA box (see Reference 32).

B. Baculovirus Expression Vector Systems

1. Advantages

Baculoviruses (especially NPVs) have been successfully used as highly efficient eucaryotic expression vectors (see reviews of References 14, 15). Baculoviruses have several characteristics that make them ideal vectors for the expression of foreign proteins, including (1) nonessential genes possessing very strong promoters that can efficiently drive foreign gene expression, (2) a double-stranded DNA genome that can be easily modified, (3) a rod-shaped viral capsid that can accommodate foreign DNA inserts of up to 10 kbp, and (4) cell lines that support viral replication. The polyhedrin gene promoter was the first to be used to drive foreign gene expression. As previously mentioned, polyhedrin is extremely highly expressed, yet nonessential for viral re...

Table of contents

• Cover
• Half Title
• Title Page
• Copyright Page
• Preface
• The Editors
• Contributors
• Table of contents
• Chapter 1: Expression of Foreign Genes in Insect Cells Using Baculovirus Vectors
• Chapter 2: Production of Baculoviruses for Insect Control Using Cell Culture
• Chapter 3: Specificity of Baculoviruses
• Chapter 4: Application of Insect Cell Culture to the Study of Bacillus thuringiensis Toxins
• Chapter 5: Large Scale Insect Cell Production
• Chapter 6: Growth and Interactions of Cells from the Insect Nervous System In Vitro
• Chapter 7: Physiological and Developmental Capacities of Insect Cell Lines
• Chapter 8: Differences in Transfection Efficiency between Drosophila Cell Lines
• Chapter 9: Intrinsic Stress Resistance of Cultured Lepidopteran Cells
• Index