Protein Expression

Our Protein Expression Services incorporate the latest developments in bacterial, baculovirus and mammalian expression technologies to assure that your recombinant protein production will be efficient and economical. We offer a number of Protein Expression Services designed to meet specific goals, ranging from expression of tagged proteins for research to production of biopharmaceuticals for clinical use. Your custom protein expression will be optimized by engineering an expression construct with a codon-optimized DNA insert, a strong promoter, an efficient ribosome binding site, a high copy number and combining it with an appropriate host. High level of protein expression also depends on stability, solubility, and folding pathway of the protein product. Accordingly, we will optimize these protein expression parameters when such optimization is necessary.

GENETIC ENGINEERING

• generation of a gene by PCR or synthesis
• subcloning into an appropriate vector
• confirmation of DNA insert by Sanger sequencing
• plasmid preparation and DNA storage
• site-directed mutagenesis

BACTERIAL PROTEIN EXPRESSION

• codon optimization for bacterial expression and gene synthesis of DNA insert
• mutagenesis of an existing construct
• generation of bacterial expression construct(s) in a selected or client-specified vector
• transformation into an appropriate host and preparation of glycerol stocks
• screening studies for the best-expressing protein variant 
• optimization of growth conditions (host, induction, media, temperature, additives) to drive either soluble or inclusion bodies expression 
• soluble vs. inclusion bodies expression assessment by SDS-PAGE/Coomassie or Western blot
• bacterial paste scale up production (read more Fermentation & Cell Culture)
• inclusion bodies wash and recovery
• recombinant protein purification from soluble lysate fraction (read more Protein Purification)
• recombinant protein purification from inclusion bodies by refolding (read more Protein Purification)

BACULOVIRUS-INDUCED INSECT PROTEIN EXPRESSION

• codon optimization for insect cell expression and gene synthesis of DNA insert
• mutagenesis of an existing construct
• subcloning of a DNA insert into a selected or client-specified baculovirus expression vector 
• virus generation, amplification and cloning by limited dilution or plaque purification 
• generation of high-titer virus stocks
• SDS-PAGE, Western, ELISA or functional expression analysis of cell lysates and conditioned media for intracellular and secreted protein products respectively
• secreted or intracellular expression in Sf9, Sf21 and HiFive insect cells
• expression optimization studies: MOI, time course, growth media
• large-scale insect cell culture for conditioned media or cell pellet production  (read more Fermentation & Cell Culture)
• recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

YEAST PROTEIN EXPRESSION

• codon optimization for yeast cell expression and gene synthesis of DNA insert
• subcloning of a DNA insert into a selected selected yeast expression vector 
• mutagenesis of an existing construct
• generation of yeast transformants
• screening for high-yielding transformants by Western, ELISA or functional expression analysis of cell lysates
• intracellular expression of membrane proteins in S.cerevisiae yeast cells
• expression optimization studies: secretion signal, host strain, media
• large-scale yeast cell culture for conditioned media or cell pellet production recombinant protein purification from conditioned media or cell pellet (read more Fermentation & Cell Culture)
• recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

MAMMALIAN PROTEIN EXPRESSION

• codon optimization for mammalian cell expression and gene synthesis of DNA insert 
• mutagenesis of an existing construct
• generation of mammalian protein expression construct(s) in a selected or client-specified vector
• endotoxin-free plasmid DNA preparation for transient or stable transfections
• small-scale trial to assess recombinant protein expression and/or to optimize transient transfection conditions
• various transient transfection reagent options
• generation of stably-transfected cell pool
• expression analysis by SDS-PAGE, Western, ELISA or functional assay
• secreted or intracellular expression in CHO, HEK293, HEK293E or client-specified cells 
• adherent or suspension cell growth options
• large-scale mammalian cell culture for conditioned media or cell pellet production  (read more Fermentation & Cell Culture)
• recombinant protein purification from conditioned media or cell pellet (read more Protein Purification)

STABLE CELL LINE DEVELOPMENT

AUXILLARY PROTEIN EXPRESSION SERVICES INCLUDE

• long-term storage for DNA, glycerol and virus stocks
• master and working cell banking

Useful Tools for a Protein Expression Project

Translate DNA to Protein Sequences
Calculate Protein Physical Properties (ProtParam)
Primer Design Tool
Protein Alignment Tool (BlastP)
DNA Alignment Tool (BlastN)

Selected Latest Developments in Protein Expression

The Ongoing Quest to Crack the Genetic Code for Protein Production.

Thijs Nieuwkoo et al., Mol Cell, 2020 Oct 15;80(2):193-209.
Abstract
Understanding the genetic design principles that determine protein production remains a major challenge. Although the key principles of gene expression were discovered 50 years ago, additional factors are still being uncovered. Both protein-coding and non-coding sequences harbor elements that collectively influence the efficiency of protein production by modulating transcription, mRNA decay, and translation. The influences of many contributing elements are intertwined, which complicates a full understanding of the individual factors. In natural genes, a functional balance between these factors has been obtained in the course of evolution, whereas for genetic-engineering projects, our incomplete understanding still limits optimal design of synthetic genes. However, notable advances have recently been made, supported by high-throughput analysis of synthetic gene libraries as well as by state-of-the-art biomolecular techniques. We discuss here how these advances further strengthen understanding of the gene expression process and how they can be harnessed to optimize protein production.
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Strategies for Enhancing Gene Expression in Escherichia coli.

Tomo Kondo & Shigehiko Yumura, Appl Microbiol Biotechnol, 2020 May;104(9):3825-3834.
Abstract
Regulation of gene expression is fundamental for cellular function. Upon manipulation of the mechanism of gene expression in Escherichia coli, various bioproducts have been developed that are valuable industrially and medically in the last four decades. To efficiently produce bioproducts, numerous molecular tools are used for enhancing expression at the transcriptional and translational levels. Our recent discovery identified a new approach that enhances the gene expression in E. coli using the gene sequence of the eukaryote, Dictyostelium discoideum. In this review, we highlight the current molecular strategies used for high-level gene expression techniques commonly utilized in basic and applied microbiology.
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Development of an Experimental Method of Systematically Estimating Protein Expression Limits in HEK293 Cells.

Yoshihiro Mori et al., Sci Rep, 2020 Mar 16;10(1):4798.
Abstract
Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.
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Construction of Gateway-Compatible Baculovirus Expression Vectors for High-Throughput Protein Expression and in Vivo Microcrystal Screening

Yanyang Tang et al., Sci Rep, 2020 Aug 7;10(1):13323.
Abstract
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies.
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Expression Vector Cassette Engineering for Recombinant Therapeutic Production in Mammalian Cell Systems

Tian-Yun Wang & Xiao Guo, Appl Microbiol Biotechnol, 2020 Jul;104(13):5673-5688.
Abstract
Human tissue plasminogen activator was the first recombinant therapy protein that successfully produced in Chinese hamster ovary cells in 1986 and approved for clinical use. Since then, more and more therapeutic proteins are being manufactured in mammalian cells, and the technologies for recombinant protein production in this expression system have developed rapidly, with the optimization of both upstream and downstream processes. One of the most promising strategies is expression vector cassette optimization based on the expression vector cassette. In this review paper, these approaches and developments are summarized, and the future strategy on the utilizing of expression cassettes for the production of recombinant therapeutic proteins in mammalian cells is discussed.
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