Expression Of Recombinant Human Protein Research Articles

Erratum: High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

An erratum was issued for:High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension. The Authors section was updated. from: Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia to Ganesh P. Subedi1 Roy W. Johnson2 Heather A. Moniz2 Kelley W. Moremen2 Adam W. Barb1 1The Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University 2Complex Carbohydrate Research Center, University of Georgia.

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human FcγRIIIa and the rat α2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression

Autologous expression of recombinant human proteins in human cells for biomedical research and product development is often hampered by low expression yields limiting subsequent structural and functional analyses. Following RNA and codon optimization, 50 candidate genes representing five classes of human proteins – transcription factors, ribosomal and polymerase subunits, protein kinases, membrane proteins and immunomodulators – all showed reliable, and 86% even elevated expression. Analysis of three representative examples showed no detrimental effect on protein solubility while unaltered functionality was demonstrated for JNK1, JNK3 and CDC2 using optimized constructs. Molecular analysis of a sequence-optimized transgene revealed positive effects at transcriptional, translational, and mRNA stability levels. Since improved expression was consistent in HEK293T, CHO and insect cells, it was not restricted to distinct mammalian cell systems. Additionally, optimized genes represent powerful tools in functional genomics, as demonstrated by the successful rescue of an siRNA-mediated knockdown using a sequence-optimized counterpart. This is the first large-scale study addressing the influence of multiparameter optimization on autologous human protein expression.

Differences in Human Growth Factors Induced by Non-Human Cell Expression.

Expression of recombinant human proteins in cells from different species, including bacteria, yeast, insect, mouse, hamster or primates, imparts species-specific changes post-translation. The impact of these species-specific differences in post-translational modifications (PTMs) can be far-reaching, resulting in effects ranging from inaccurate quantitation of proteins in serum to in-vivo immunogenicity (Porter, J Pharm Sci 90: 1–11, 2001) or even autoimmunity to the patient's own native protein (Ryan et al., Int Immunopharmacol 4:647–55, 2006). Currently, most human proteins are still produced from bacteria, including several of the World Health Organization international growth factor standards. For example, present-day clinical grade recombinant human growth factors such as G-CSF, IL-4 and GM-CSF are manufactured from bacteria. The expression of human proteins that naturally exhibit a high degree of glycosylation, such as GM-CSF, in bacterial cells has major implications for immunoassays and therapeutics. Since bacterial cells do not glycosylate human proteins, exposure of cryptic or masked epitopes, normally buried on the native protein, can result in generation of antibodies that bind falsely in immunoassays and can cause reduced potency or adverse reactions in patients. The expression of recombinant human proteins in non-human mammalian cells can result in non-natural epitopes due to the addition of non-human sugar structures. We have expressed several human growth factors in human cells and have demonstrated differences in their immunoreactivity patterns when compared to the same recombinant proteins expressed in non-human cells. These changes in the biological properties of the proteins are likely related to differences in glycosylation and other PTMs unique to proteins expressed in human cells. We have demonstrated that expression of human growth factors in human cells may have significant benefits for the accuracy of native human protein assays and the therapeutic efficacy of human growth factors.

Expression and Purification of Recombinant Human SPARC Produced by Baculovirus

SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40), a matrix-associated protein, disrupts cell adhesion and inhibits the proliferation of many cultured cells. We report the expression of recombinant human protein (rhSPARC) in a baculovirus expression system. This procedure routinely yields ∼1 mg of purified protein per 500 ml of culture supernate. rhSPARC produced by insect cells migrates at the appropriate molecular weight under reducing and nonreducing conditions. The rhSPARC purified from insect cell media appeared structurally similar to SPARC purified from mammalian tissue culture by the criterion of circular dichroism. In addition, a series of anti-SPARC and anti-SPARC peptide antibodies recognized insect cell rhSPARC. We also show that rhSPARC produced in this system is glycosylated and is biologically active, as assessed by inhibition of endothelial cell proliferation and induction of collagen I mRNA in mesangial cells. Significant amounts of rhSPARC can now be generated in the absence of contaminating mammalian proteins for structure/function assays of SPARC activities.

CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins

Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.