Protein expression lectures on higher eukaryotes               David Nelson    Mar. 25, 1997

Done.   Last modified 8:45AM Mar 25
to be covered:

baculovirus system (insect cells)
ecdysone inducible mammalian system
Sindbis virus system (many cell types)
Xenopus oocyte injection
Plant expression (The blue rose project of Florigene Pty. Ltd.)
Vaccine production in beans, potatoes and tobacco
Saffron, the worlds most expensive spice
Human proteins in cow's milk (PPL, from those people that brought you Dolly the sheep)

	Today we are going to cover a variety of expression systems for higher eukaryotic 
cells.  The best known of these is the baculovirus system for overexpression of a protein in 
insect cells or whole insect larvae.  Very high levels of expression are possible with this 
technique.  The advantages of this method over E. coli are the proper post translational 
modifications that can be achieved, including glycosylation, phosphorylation, myristolation 
and palmitolation.   (Glycosylation is probably not exactly like mammalian glycosylation, 
this is being studied).  

The baculovirus system

	The baculovirus system requires homologous recombination inside transfected 
insect cells.  This recombination takes place between a linearized version of the baculovirus 
genome with part of an essential gene missing and a transfer vector carrying the needed 
missing piece and the desired gene.  The gene to be overexpressed has to be cloned into the 
transfer vector, where it is under the control of the strong polyhedrin promoter that 
normally controls formation of the major baculovirus protein.  The transfer vector is small 
and easy to manipulate, like most cloning vectors.  The baculovirus genome is larger and 
more difficult to modify, so the cell recombines them both to create a complete baculovirus 
genome with your favorite gene going along for the ride.  In this arrangement, the viral 
genome is the same in all transfections, while the transfer vector can be different each time.  
The size of the inserted gene can be large.  The upper limit is not known.  Baculovirus 
expression can splice introns, but the results will probably be better if a cDNA is used.

	When this system was first developed, the transfected cells that made intact 
recombinant virus had to be identified by looking at the cells under a microscope to identify 
an occlusion body negative viral plaque.  The occlusion bodies are formed form the 
polyhedrin gene product that acts as a matrix for the viral particles.  There is a subtle 
difference in appearance of the occlusion body positive and negative infected cells.  It was 
necessary to pick the recombinant plaques to purify the recombinant virus so more cells 
could be infected.  After several rounds of plaque purification, all the progeny virus should 
be identical and contain the inserted gene.  This was not an easy task.  To improve the 
detection of the recombinant virus in the transfected cells, lacZ has been added to the 
system.  Part of lacZ is on the linearized genome, and an overlapping part of it is in the 
transfer vector.  when recombination occurs it  has to take place between the two parts of 
lacZ.  This restores active beta galactosidase to the recombinant cells and turns the cells 
blue.  

	The virus used is called the Autographa californica nuclear polyhedrosis virus 
(AcMNPV).  The insect cells are either Sf9 cells or Sf21 cells, both from Spodoptera 
frugiperda (fall army worm) ovary cells.  When whole larvae are infected, army worms are 
used.  

	Refinements have been made to the system so that Histidine tags can be added at the 
N-terminal of your protein.  These also have an enterokinase proteolytic cleavage site after 
the His-tag so it can be removed after purification on nickel chelate resins.  The His-tag 
vectors come in three varieties, one for each reading frame, each has 10 restriction sites in 
the polylinker region.  

	If you want to have your protein secreted, a honey bee mellitin signal sequence can 
be fused in frame with your gene.  The product will then be targeted to the ER for the 
secretion pathway.   The signal sequence will be cleaved off in the processing for secretion, 
leaving a wild type native protein.

	Invitrogen makes all the needed items for baculovirus expression.  Their kit is 
called MaxBac 2.0. Clonetech also makes baculovirus kits called BacPAK. The Invitrogen
baculovirus manual and their other manuals as well are available to download on the web in 
Acrobat PDF format from http://www.invitrogen.com 

Ecdysone inducible expression in mammalian cells

	Ecdysone is an insect steroid hormone that is involved in insect molting.  Ecdysone 
binds to a heterodimeric receptor composed of the ecdysone receptor (VgEcR) and USP 
(ultraspiracle).  Each of these proteins has a DNA binding domain that is unique.  ecdysone 
binding causes the dimeric receptor to bind its target DNA sites and activate transcription of 
genes involved in molting.  

	This system has been modified to make it bind to sequences on a vector that have 
been engineered to be unique in mammalian cells.  The VgEcR receptor has had the VP16 
transactivation domain (used in two hybrid systems) added to it to make it a better activator 
of transcription.  In addition, the ecdysone receptor's DNA binding domain has been 
altered to recognize a hybrid response element.  One half of the response element is the 
normal ecdysone response element.  The other half is the glucocorticoid response element.  
This hybrid response element is called (E/GRE).  Instead of using USP that is specific for 
insects, the mammalian homolog of USP, the retinoid X receptor (RXR), is used for the 
second subunit of the heterodimer.  With all this modification, the only site the modified 
ecdysone heterodimer can recognize is a synthetic sequence that is only found on a vector 
upstream of the gene to be expressed.  In fact the vector has 5 of these modified E/GRE 
elements in front of the minimal heat shock promoter.

	To make this system work in mammalian cells, two vectors have to be transfected 
into the cells.  One contains both subunits of the receptor, and the other contains the gene 
of interest with the E/GRE elements upstream.  An analog of ecdysone called muristerone 
A is used.  Each vector has a different resistance gene, one is for neomycin resistance and 
the other is for Zeocin resistance.  (Zeocin is a drug similar to bleomycin that intercalates 
into DNA and cleaves it).  With both vectors in a cell, addition of muristerone A causes a 
greater than 200 fold induction of expression of the target gene over basal levels.  

The Invitrogen catalog claims that the ecdysone expression system is the only truly 
inducible mammalian expression system.  The response is also dose dependent so you can 
control the level of expression with the concentration of muristerone A.

Sindbis virus as a vector for expression

	Sindbis virus expression allows transient gene expression in a variety of eukaryotic 
cell lines.  These include mammalian, avian, reptilian and insect cells (mosquito and 
Drosophila).  It is often desirable to test your protein in different cell types, since unique 
sets of proteins are expressed in each cell type and different responses may be expected.  
The highest levels of expression are found in BHK (baby hamster kidney) cells.  

	The first task in expressing a gene in this system is to clone your gene into a 10kb 
plasmid called pSinRep5.  The gene is placed downstream of a promoter called the 
subgenomic promoter.  Also on the same plasmid are four genes required to replicate the 
virus RNA in the cells.  The vector does not contain genes for the structural proteins for 
making the virus particle, so these must be added by a defective helper DNA if you want to 
package your gene into virions to infect other cells.  

	Once the gene is in this vector, the vector is linearized by one of three rare cutting 
restriction enzymes, just beyond the poly A tail of your gene.  The linearized DNA is 
reverse translated into RNA that is capped.  This is done in vitro.  The capped and 
polyadenylated linear RNA is transfected into BHK cells where it is translated to make the 
four non-structural proteins of Sindbis.  These replicate both the genomic RNA (the 
nonstructural genes) and the subgenomic RNA (your gene).  This subgenomic RNA 
becomes the most abundant message in the cell, so your protein is expressed at fairly high 
levels.  This is not like the E. coli system where you could achieve 50% of total cell 
protein.  Here you will do well to get 1-3%.  

	To infect other types of cells, pseudovirions have to be made that encapsulate the 
linear RNA that you originally added to the cells.  This is done by adding defective helper 
RNA that codes for the virion proteins.  Together, both act to form virions and only the 
original RNA is packaged in the heads, because it carries a packaging signal.  The defective 
helper does not.  Viral particles bud into the medium and these can be harvested to infect 
other types of cells.  The virions can only undergo one round of infection, because they 
lack the helper RNA and cannot make virions.

Xenopus oocyte expression

	I am only going to introduce you briefly to the Xenopus oocyte as a system for 
expression.  There are two different experimental approaches that can be taken with 
Xenopus.  The first is to inject DNA into the egg nucleus and get transcription to occur, the 
other is to inject RNA into the cytoplasm and get translation.   The injection is done with a 
micromanipulator and a micropipet.  Dr. Taylor actually does this in his lab, so I refer you 
to him if you want to know the details of how it is done.  

	The oocyte is a useful system to produce proteins for specific sensitive bioassays.  
The function of mutant ion channels made in very small quantities can be measured by 
patch clamp methods.  Even multiple subunit membrane proteins like the acetylcholine 
receptor can be assembled into a functional unit if all the RNAs can be added.   The oocyte 
then becomes the poor man's transgenic animal.  The oocyte system is not made for large 
scale protein production, but it is suitable for more specialized purposes.

The Blue Rose Project

	Florigene, a company based in Australia is interested in expressing proteins in 
flowers, not to make the protein, but to engineer in a pathway for flower pigments.  For 
centuries, a blue rose has been the subject of fiction.  It was mentioned in the Arabian 
Nights.  There is no such thing as a blue rose however, because the key enzyme in the 
pathway to blue or purplish pigments is lacking in the rose family.  This enzyme is a 
flavonoid 3'5' hydroxylase.  This enzyme acts on anthocyanins that are already 
hydroxylated at the 3' and 4' positions to add a third hydroxyl at the 5' position of the 
anthocyanin B ring.  This pigment is bluish or purplish in color and its specific absorption 
properties can be modified by pH, metal ions and copigments.  

	Florigene has developed methods to transform genes into carnations, 
chrysanthemums and roses.  They have cloned the genes for flavonoid 3'5' hydroxylase 
from petunia flower petals and expressed these genes in carnations.  This has led to 
purplish colored carnations.  They have not got all the additional factors worked out yet to 
get a true blue color expressed, but they are working on it.  In addition, they are 
transforming the genes into chrysanthemums and roses.  The estimated world wide market 
for a blue rose is in the 3-5 billion dollar a year range, so it is worth the initial trouble to 
engineer this pathway into roses.  

Vaccine production in beans and potatoes

	The new trend in vaccine production is to make subunit vaccines based on the 
immunogenic proteins and even specific linear epitopes of proteins from a disease causing 
organism.  One of the latest developments has been to engineer a linear epitope from an 
animal virus into the coat protein of a plant virus.  When the plant host was infected the 
modified virus was made with the epitope of the animal virus on the surface of the plant 
viral particles.   In this case the plant was the black-eyed bean and the virus was cowpea 
mosaic virus (Nature Biotechnology 15,248-252 1997).  The recombinant virus particles 
were used to inject mink.  These immunized animals were protected against the mink 
enteritis virus which was the source of the epitope.  This demonstrated that the plant 
derived chimeric virus was a true vaccine, even though it only contained 17 amino acids 
from the mink enteritis virus.  One relevant fact about this epitope is that it is exactly the 
same in a related feline virus and a dog virus, so one vaccine might be able to protect three 
different animals from three different viruses.    

	Another approach to plant derived vaccines is to make transgenic plants, rather than 
chimeric viruses.  This has been done with potatoes.  The immunogenic protein was 
expressed in the potato and mice that ate the potatoes developed antibodies against the 
antigen.  This may be a case of an edible vaccine that could be used in tropical areas where 
refrigeration is not available.  Transgenic tobacco has been made that expressed hepatitis B 
surface antigen which was also shown to be antigenic.  Vaccine production in tobacco may 
be a justifiable industry for tobacco companies to consider.  

Saffron, the worlds most expensive spice

	Saffron is a spice made from the stigmas of the crocus flower Crocus sativus.  It is 
grown in Spain in La Mancha, in Greece and in Iran.  The best saffron comes from La 
Mancha and it is identified as Spanish Saffron.  I have brought a bottle to pass around.  It 
is so valuable, that a regulatory body provides a seal authenticating La Mancha saffron, to 
distinguish it from Greek or Iranian saffron.  The bottle going around the room has 1.7 
gram of saffron and it cost $14.  This works out to be about $3700 per pound or $8200 per 
kg.Saffron has about 1% of a volatile oil consisting of picrocrocin (glucose plus safranal) 
and crocin.  As you might expect, there is an interest in the genetics of saffron production.  
The lab plant Arabidopsis has a gene called superman that doubles the number of male 
flower parts.  It seems likely that a similar gene might be found that could double the 
number of stigmas (the female flower parts).  If this could be done in the crocus that would 
double the yield.  Also, the pathways involved in synthesis of the flavors in saffron might 
be engineered into yeast or bacteria to make the spice without the plant.  Since the flavor 
might be due to a complex mixture of chemicals, this last approach may not be easy.

Human proteins secreted in cows milk

	PPL Therapeutics, the company that funded Ian Wilmut, the man who cloned Dolly 
the sheep, has been making transgenic cows that produce human alpha lactalbumin.  This is 
a normal protein of milk, but it is now possible to get the human version in cow's milk.  
The industry sees potential for improved infant formulas and specialty diets.  Engineering 
of an alpha lactalbumin without phenylalanine could help to make nutritional supplements 
for phenylketonurics.  

Industrial enzymology

	Companies are arising now with the purpose of isolating, expressing and marketing 
novel and useful enzymes.  One company called Recombinant Biocatalysis offers access to 
the worlds extremophiles and their enzymes.  This is intended for commercial application 
of enzymes to industry.  They claim to have discovered 175 new enzymes that they call 
Clonezymes.  They package these in groups of 3-10 that have related activities for clients to 
screen for useful candidates for a given process.  The company will help customers by 
evolving any of their enzymes to specifications of pH and temperature optima.  This is 
done by multiple rounds of random mutagenesis followed by screening to select those 
mutants that come closest to having the desired properties.  The process is repeated until the 
goal is achieved.  This is called DirectEvolution.  http://www.biocat.com