Biotechnology of drugs– Expression of
recombinant proteins in eukaryotic cells
Doc. RNDr. Jan Hošek, Ph.D.
hosekj@pharm.muni.cz
Department of Molecular Pharmacy
FaF MU
Aims of transfection of animal cells
1. Gene transfer into animal cells
• searching for genes, learning their function and regulation and studying the
phenotype (e.g. differentiation processes and their disorders)
• the study of protein interactions
• formation and purification of foreign proteins
2. Preparation of transgenic animals
• the study of the functioning of genes within the whole organism
• models for the study of genetic diseases
• preparation of animals with better useful properties
• searching for options for gene therapy
• creation of foreign proteins (gene pharming)
Insect cell expression systems
• Host cells derived from
– Spodoptera frugiperda → Sf9 cells –
transient expression
– Trichoplusia ni → High Five cells –
transient expression
– Drosophila melanogaster → S2 cells –
stable expression
– They can grow adherently or in
suspension
• Baculovirus vector
➢ The most common member is Autographa
californica multiple nuclear polyhedrosis
virus (AcMNPV) parasitizing members of
the order lepidoptera (Spodoptera
frugiperda and Trichoplusia) → a wide
range of hosts
➢ Large rod-shaped dsDNA viruses
infecting insects
➢ Viral particles are enveloped by a protective matrix consisting of the
protein polyhedrin - it allows survival in the external environment and
efficient spread between cells, in in vitro conditions are not required
➢ The pPolh promoter allows expression of polyhedrin or
recombinant protein - up to 50% of the total protein at the end of
the baculovirus cycle
Baculoviruses
Baculovirus expression vectors I.
DOI: 10.1016/S0076-6879(09)63014-7
• Gene of interest under pPolh promoter
• GOI on plasmid → homologous
recombination to AcMNPV DNA
• Low success rate of recombination
(approx. 0.1%)
• More complicated search for
recombinant (polyhedrin-negative)
viral plaques
• Use of linearized (non-replicating)
AcMNPV DNA → recombination
success rate 10-20%, BakPAC system
up to 95%
DOI: 10.1016/S0076-6879(09)63014-7
Baculovirus expression vectors II.
• Use of transposition instead of
homologous recombination
• Transposition takes place in E. coli,
which carries a "helper" plasmid
encoding the transposase
• Recombination success rate 100%
DOI: 10.1016/S0076-6879(09)63014-7
➢ binary vector that can replicate in
Escherichia coli and insect cells
(contains complete baculovirus
genome (no PH), low-copy origin of
replication from F plasmid and att
site for T7 transposon)
➢ carries resistance to kanamycin
➢ carries the lacZ gene, so colonies
transformed by it grow blue in the
presence of IPTG
➢ the recombinant gene is introduced
into it by transposition from another
vector, the transposition is directed
to lacZ, the resulting colonies grow
white
Bacmid
http://strubiol.icr.ac.uk/extra/baculovirus/introduction.html
transponase
https://www.creativebiomart.net/baculovirus-insect-cell-expression-systems.htm
Construction of recombinant Bacmid
regions flanking the
polyhedrin gene
by incorporating the plasmid into the
baculovirus genome (2x CO) a shuttle
vector is created (E. coli + insect cells)
the transposition function of the helper
plasmid will enable the transposition of
the stretch of the donor plasmid
containing the GOI, which is under the
control of the baculovirus promoter and
terminator (p and t)
the recombinant bacmid has a disrupted
lacZ' gene; E. coli cells containing
recombinant bacmid are unable to form
functional β-galactosidase (white
colonies)
➢ suitable for the production of high levels of
recombinant proteins (up to 1000 mg/mL)
➢ correctly post-translationally modified
(folding, formation of S-S bridges,
oligomerization, glycosylation, acylation,
proteolytic cleavage)
➢ biologically active and functional recombinant
proteins
➢ the recombinant gene is inserted into regions
of the viral genome that are not required for
virus replication
Basic features of the insect system I.
➢ recombinant baculovirus loses one of the
dispensable genes (polh, v-cath, chiA ...), which is
replaced by a recombinant gene
➢ the recombinant protein is expressed in insect cell
cultures or insect larvae (not so common)
http://www.ent.iastate.edu/dept/faculty/bonningb/files/images/zebracaterpillar.jpg
Basic features of the insect system II.
➢ High levels of gene expression, especially for intracellular
proteins
➢ Recombinant proteins are mostly soluble, post-translationally
modified and easy to isolate, the content of parental proteins
is low, expression takes place in the late stages of infection
➢ Insect cells grow well in suspension cultures - easy transfer to
bioreactors
➢ Heterooligomeric proteins can be expressed using
simultaneous infection with two or more viral vectors or a
vector with multiple expression cassettes
➢ Baculoviruses have a limited spectrum of invertebrate hosts safe
technology
Advantages of the insect express system
Glycosylation of proteins in insect cells
• Insect cells have different glycosylation
compared to mammalian cells
➢ Reduced biological activity
➢ Immunogenicity / allergic reaction
• Use of insect cells transfected with
mammalian glycosyltransferases
doi:10.1038/nbt1095
DOI:10.1016/S0065-3527(06)68005-6
doi:10.1021/acs.iecr.8b0098
doi:10.1021/acs.iecr.8b0098
Transient expression in insect cells
• Mainly Sf9 cells are used
http://dx.doi.org/10.5772/67849
doi:10.1021/acs.iecr.8b0098
Use of transient expression
http://dx.doi.org/10.5772/67849
doi: 10.1002/biot.201400438
Stable expression in insect cells
• Mainly S2 cells → recombinant rS2 cells
• The gene is integrated into the nuclear DNA
• Stable expression, high batch homogeneity
• The possibility of using perfusion cultivation systems
http://dx.doi.org/10.5772/67849
Use of stable expression
http://dx.doi.org/10.5772/67849
Vectors for transfer into mammalian cells
1. Plasmid vectors - the plasmid does not replicate, it is rarely
incorporated into the genome of the cell
• prokaryotic plasmid + eukaryotic transcription unit + selection marker
• they are used to select transfected cells during co-transfection and to monitor
transient gene expression
2. Viral vectors - shuttle vectors, replicating in host cells
• part of the bacterial vector + sequence of eukaryotic viruses + selection
marker
• vectors derived from SV40, bovine papillomavirus, EBV, retroviruses,
baculoviruses, vaccinia virus, adenoviruses, etc.
• they are used to monitor stable or transient gene expression and to obtain
recombinant proteins in large quantities
General structure of a mammalian expression vector
• Promoter
• Enhancer of
transcription
• polyA sequence
• Selection marker
• Replication origin
https://doi.org/10.1007/s00253-020-10640-w
Mammalian vector promoters
• The most common promoters for expression in mammalian cells:
– human cytomegalovirus enhancer/promoter (hCMV)
– Simian virus 40 early promoter (SV40E)
– CMV enhancer/chicken β-actin promoter (CAG)
– human elongation factor-1α (hEF-1α)
– Elongation factor-1α of Chinese hamster (CHEF-1α)
• Artificially created SCP1 (super core promoter 1) promoter → 3x faster than CMV
promoter
https://doi.org/10.1007/
s00253-020-10640-w
Viral constitutive promoters
• High level of GOI expression
• Viral promoters are often only active during a specific
phase of the cell cycle (e.g. CMV is active during S
phase)
• High production of the target protein inhibits cell growth
and can also cause apoptosis
• Viral promoters are susceptible to epigenetic silencing
Inducible promoters
• They are not cell cycle dependent
• Induction of protein expression at the peak of the exponential phase
of growth will ensure maximum yields
• IPTG-inducible lac operon
• Tetracycline resistance repressor operons Tn10 (tetracycline
resistance repressor operons) – Tet-Off/Tet-On
Other regulatory sites
• PolyA sequence
– in 3‘-UTR of terminator
– polyA sequence from the SV40 virus –
increased resistance to nucleases
https://doi.org/10.1016/j.molcel.2020.03.022
Other regulatory sites
• Introns
– inserting an intron or untranslated exon before the start codon increases the efficiency of
mRNA transport from the nucleus to the cytoplasm and prolongs its half-life
https://genomebiology.biomedcentral.com/articles/10.1186/gb-2002-3-3-reviews0004
Other regulatory sites
• IRES
– internal ribosome entry site
– it is inserted between the reading frames of bi/poly-cistronic mRNAs
– ensures the expression of multiple genes from a single promoter
– translation of the gene down-stream the IRES is lower than up-stream the IRES
https://doi.org/10.1016/B978-0-12-394447-4.30042-6
https://doi.org/10.3390/biomedicines10081865
Other regulatory sites
• Furin-2A sequence
– small self-cleaving peptides
– they are inserted between the two ORFs instead of the IRES
– will ensure equal expression of both genes
https://commons.wikimedia.org/wiki/File:2A_peptide_Working_Mechanism.jpg https://doi.org/10.3390/pharmaceutics11110580
Selective markers
Formation of multimeric proteins
• Preparation options :
1. cloning the genes for the subunits separately and then joining them in vitro –
low efficiency
2. gene cloning in two vectors in one cell - protein assembly in cellulo - high
efficiency
• Possible complications:
– Loss of one of the vectors, the active protein is not made
– Different copy number of vectors, one of the subunits is in excess, the
resulting product is not enough
• Solution: Cloning genes in a bicistronic expression vector
Two-vector expression system in a single cell
• Loss of one of the vectors, the
active protein is not made
• Different copy number of
vectors, one of the subunits is
in excess, the resulting
product is not enough
Dimeric protein
Two genes on one vector
Expression vector with two cloned genes
encoding heterodimer subunits
Bicistronic expression vector for cloning
genes encoding heterodimer subunits
both genes
are
transcribed
and translated
separately
one transcript
(bicistronic)
translated into
two protein
units
Retroviral vectors
• They infect a wide range of animal species and various types of
human cells
• Infection leads to the integration of the viral genome into the genome
of the host cell - the place of integration is arbitrary (preferably in
transcriptionally active chromatin)
• Retrovirus infection does not result in cell death, it often leads to
constant production of new virions
• A disadvantage is the ability of retroviral vectors to activate the
transcription of genes adjacent to their insertion sites
Vectors derived from SV40 virus
• Simian virus 40 – polyomavirus attacking
monkeys and humans
• genome = 5.2 kb circular dsDNA
• life cycle:
– in permissive cells (monkeys) – lytic cycle
– in non-permissive cells (mouse, hamster) –
integration into the genome, cell transformation
• Construction of vectors:
– replacement of the early or late region with
foreign genes, complementation of missing
functions with a helper virus or helper cells (COS)
COS cells
• Fibroblast-like cells derived
from monkey kidney tissue
• COS = CV-1 (simian) in Origin,
and carrying the SV40 genetic
material
• Defective SV40 virus
integrated into the genome cannot
replicate independently,
but makes all proteins
• Replication of recombinant
SV40 viruses or plasmids with
oriSV40
• mainly transient expression
https://doi.org/10.1016/S0167-7306(03)38005-6
Mammalian cells used
• Most commonly used CHO cells (60-
70% of recombinant
biopharmaceuticals on the market)
– mainly stable expression
– grow in suspension
– protein production up to 10 g/L
• Sp2/0 and NS0 mouse myeloma,
YB2/0 rat myeloma and baby
hamster kidney (BHK) cells
• Human lines PER C6 and CAP
– ideal post-translational modification
→ low immunogenicity of therapeutic
proteins
doi: 10.1007/978-3-319-52287-6_29
doi:10.1007/978-3-319-52287-6_29