Engineering of Fusion Proteins

Particularly for bacterial expression, fusion to prokaryotic proteins such as the maltose binding protein (MBP),5 glutathione S-transferase (GST),6 and bacteriophage PRD1 membrane protein7 has produced favorable results because the fusion partner has been demonstrated to improve yields and provide more stable proteins.

Cell-free

Lysates

E. coli Wheat germ |1 day PCR product

|1 day Expression 12 days Scale-up

TOTAL 4 days

Prokaryotes Eukaryotes

Bacteria Yeast

E. coli

H. salinarium L. lactis

Subcloning

12 days Expression 12 days Scale-up

TOTAL 11 days

S. cerevisiae S. pombe P. pastoris

7 days

Subcloning

7 days

Clone selection

^2 days

Expression

^2 days

Scale-up

TOTAL 18 days

Insect

7 days

Subcloning

TOTAL 21 days

Mammalian

Transient Stable

7 days

Subcloning

3 days

10 days

Baculovirus production

12 days

Expression

12 days

Scale-up

Transient expression

12 days

Scale-up

TOTAL 12 days

Stable cell line

Viral

Adenovirus SFV

14-21

days

Subcloning

12 days

SFV production

expression 12 days

|2 days AdV Scale-upexpression

3 days

AdV production

2 days

Stable expression

12 days

Scale-up

TOTAL 12 days

2 days

Scale-up

TOTAL 14 days

Baculovirus

7 days

7 days

TOTAL 25-32 days

FIGURE 8.1 Procedure and time requirements for recombinant GPCR production in various expression systems. Key steps in different processes are shown.

8.1.4 Receptor Production for Structural Biology Applications

In this chapter, the various expression systems generally applied for overexpression of recombinant proteins are described. Most of these systems have also been used for GPCR expression and their special properties and achievements are presented below. Recombinantly expressed GPCRs provide the material for assays to monitor direct ligand binding activity as well as functional coupling to G proteins; both types of assays are used widely for drug screening programs.

As a general rule, high expression levels are desired for binding assays because high receptor densities in isolated membranes allow screening of a larger number of compounds. In contrast, functional assays seem to work better for lower receptor concentrations since the ratio of GPCRs to G proteins becomes more favorable. Because the drug discovery aspects of GPCRs are described elsewhere, the main focus of this chapter is on receptor production for structural biology applications.

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