Identifying opportunities in cell engineering for the production of ‘difficult to express’ recombinant proteins

There is a growing demand for production of recombinant proteins of many structural varieties in mammalian expression systems, either as therapeutics or for protein characterisation. However, certain recombinant proteins are “difficult to express” in mammalian expression systems requiring extensive cell line and process optimisation which, as a result, can have significant consequences for drug development processes. The Tissue Inhibitors of Metalloproteinase (TIMP) protein family, TIMP-2, -3 and -4, are naturally secreted proteins that share significant structural homology (~50% identity and ~70% similarity in amino acid sequence), but show profound differences in secretion in mammalian expression systems. Computational sequence analysis of the TIMPs shows areas of significant amino acid difference mainly locating to flexible loop regions. This study has investigated the molecular mechanisms that selectively restrict expression of recombinant proteins of extensive sequence similarity. The loci of the molecular steps that limit successful expression have been defined by quantitative real-time polymerase chain reaction, proteomic analyses, cellular fractionation and immunofluorescence microscopy. All three TIMPs were readily detectable at mRNA and protein level within the cell but only TIMP-2 was secreted effectively into the culture medium. Analysis of protein localisation showed intracellular protein for all three TIMPs, mainly co-localised in the organellar and cytoskeleton fractions. In addition, immunofluorescence microscopy showed all three TIMPs to be detectable within the endoplasmic reticulum. TIMP-3, which was not secreted, was detected within the cell in both expected glycosylated and non-glycosylated forms. Treatment of intracellular TIMP-3 with glycosidases suggests the presence of an immature high mannose glycoform. Knockout of the TIMP-3 glycan site did not result in secretion. These data suggest that the post-translational processing of poorly expressed TIMPs limits transit through the secretory pathway. To overcome this challenge, cell engineering of limiting secretory pathway components could enhance production of these “difficult to express” recombinant proteins

Enrollment of adolescents and young adults onto SWOG cancer research network clinical trials: A comparative analysis by treatment site and era.

BackgroundFew adolescents and young adults (AYAs, 15-39 years old) enroll onto cancer clinical trials, which hinders research otherwise having the potential to improve outcomes in this unique population. Prior studies have reported that AYAs are more likely to receive cancer care in community settings. The National Cancer Institute (NCI) has led efforts to increase trial enrollment through its network of NCI-designated cancer centers (NCICC) combined with community outreach through its Community Clinical Oncology Program (CCOP; replaced by the NCI Community Oncology Research Program in 2014).MethodsUsing AYA proportional enrollment (the proportion of total enrollments who were AYAs) as the primary outcome, we examined enrollment of AYAs onto SWOG therapeutic trials at NCICC, CCOP, and non-NCICC/non-CCOP sites from 2004 to 2013 by type of site, study period (2004-08 vs 2009-13), and patient demographics.ResultsOverall, AYA proportional enrollment was 10.1%. AYA proportional enrollment decreased between 2004-2008 and 2009-2013 (13.1% vs 8.5%, P < .001), and was higher at NCICCs than at CCOPs and non-NCICC/non-CCOPs (14.1% vs 8.3% and 9.2%, respectively; P < .001). AYA proportional enrollment declined significantly at all three site types. Proportional enrollment of AYAs who were Black or Hispanic was significantly higher at NCICCs compared with CCOPs or non-NCICC/non-CCOPs (11.5% vs 8.8, P = .048 and 11.5% vs 8.6%, P = .03, respectively).ConclusionNot only did community sites enroll a lower proportion of AYAs onto cancer clinical trials, but AYA enrollment decreased in all study settings. Initiatives aimed at increasing AYA enrollment, particularly in the community setting with attention to minority status, are needed

Use of a ‘molecular tug’ to overcome limitations in the production of ‘difficult to express’ recombinant proteins

In recent years there has been an increased drive towards the production of recombinant proteins in large amounts using rapid cell culture processes. Mammalian expression systems such as Chinese Hamster Ovary (CHO) cells have remained the preferred choice for large-scale recombinant protein production (Walsh, 2014). However, in mammalian cells certain recombinant targets can prove to be ‘difficult to express’ and require extensive upstream process optimisation which can have a negative impact on industrial processes. This study has investigated the molecular mechanisms that are responsible for poor recombinant protein production. Model proteins from the Tissue Inhibitors of Metalloproteinase (TIMP) family, TIMP-2, TIMP-3 and TIMP-4, were subject to detailed study to characterise the molecular mechanisms that limit production of recombinant proteins with high sequence homology (Hussain et al., 2017). TIMP-2, -3 and -4, share significant sequence/structural homology (Douglas et al., 1997, Garcia et al., 2012), but show differences when produced in a transient CHO expression system (Hussain et al., 2017) . A systematic screen of the protein expression pathway showed all three TIMPs were detectable at the mRNA and protein level within the cell but only TIMP-2 was secreted in significant amounts into the culture medium. Analysis of the intracellular protein suggested the post-translational processing of poorly expressed TIMPs was limiting. A protein engineering approach was employed to overcome challenges in the production of these ‘difficult to express’ TIMP proteins. This approach involved the attachment of a furin-cleavable pro-sequence from a secretory growth factor to recombinant targets. The pro-sequence was predicted to act as a ‘molecular tug’ to aid transit through the protein expression pathway and/or promote correct post-translational processing. Initially, the furin-cleavable pro-sequence was added to TIMP-3 (non-secreted), which resulted in secretion of TIMP-3, however incomplete processing of the pro-sequence was observed. The protein engineering approach was optimised further and applied in combination with cell engineering (furin overexpression) to TIMP-4 (poorly secreted), which was also successfully detected in significantly higher amounts in the culture medium (Hussain et al., 2017). Together, the described protein engineering approach presents a novel strategy to increase the production of ‘difficult-to-express’ recombinant targets. References: Douglas, D. A., Shi, Y. E. & Sang, Q. X. A. 1997. Computational sequence analysis of the tissue inhibitor of metalloproteinase family. Journal of Protein Chemistry, 16, 237-255. Garcia, M. P. S., Suarez-Penaranda, J. M., Gayoso-Diz, P., Barros-Angueira, F., Gandara-Rey, J. M. & Garcia-Garcia, A. 2012. Tissue inhibitor of metalloproteinases in oral squamous cell carcinomas - A therapeutic target? Cancer Letters, 323, 11-19. Hussain, H., Fisher, D. I., Abbott, W. M., Roth, R. G. & Dickson, A. J. 2017. Use of a protein engineering strategy to overcome limitations in the production of Difficult to Express recombinant proteins. Biotechnology and Bioengineering, 114, 2348-2359. Walsh, G. 2014. Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32, 992-1000

Prospectus, November 14, 1984

YOUR COURAGE, DEDICATION AND SACRIFICES ARE NOT FORGOTTEN; Parkland Veterans Honored; Women Veterans receive deserved recognition; Landslide of volunteers hoped for; Recognition needed all year; Blood Drive November; Best Sellers in the Library; PC Happenings; Original performance at Parkland; How to Live to be 1000 is retirees topic; Gallery hosts touring exhibit; EMT refresher workshop scheduled; Parkland, GM cooperate; Student job prospects look great; Man on Street...Question: Did you vote on Tuesday, and whatever you answer, why?; Creative Corner...especially for you!; Doom story...The cataclysm begins; Fluffy White Clouds; Nursing on a psychiatric floor; Ember of Empathy; With Feeling; Hurry Monday!; Contradiction versus deception; the eve of destruction; Homo Sapiens; Essence; Straub has not even begun to hit his stride; Good music sometimes underrated; Anothe Bowie hit; Classifieds; Britter\u27s Knights devastate USA Adidas 113-55 in opener; Lady Cobras finish spectacular season with 39-9-1 clipping; Knights down Nonames 100-30; Cobra cagers have flexibility and depth for \u2784-\u2785 campaign; Stater, Phillips supply one-two punch for Cobrashttps://spark.parkland.edu/prospectus_1984/1004/thumbnail.jp

Prospectus, November 28, 1984

SPEAKING COBRAS NAMED COMMUNITY COLLEGE TEAM OF THE YEAR; Central Illinois Vietnam Veterans; Security reports thefts; Parkland election survey told; \u27There are 25 crucified saviors\u27; PC Happenings; Board of Trustees re-elect officers; Who\u27s Who Award nominations listed; Parkland Choral Union to perform \u27Messiah\u27; \u27Messiah\u27 comes again; Life Science Division has open house; Classifieds; Scott advocates negotiation versus doomsday; Creative Corner...especially for you!; Doom Story...The Final Conflict; Through another man\u27s eyes; and that is all; affair; your last veil; living together; Jesus Face; Jesus Died; This Isn\u27t It; wanting; being in love; On Being Forced to Play Backgammon After a Week in the Wilderness; only one; Searls Scouts; Talent scouts looking for fresh talent to work at Busch Gardens in 1985; King\u27s newest not his best; Parkland Community Band performs; Prompters cheer IRT; Cotton releases new solo; Original theatre production at Parkland Written, directed and produced by Lu Snyder; Carol sing is Sunday at UI; High School Quiz Bowl to be broadcast; Howard Player of the Week; Parkland loses on last-second shot; High School Notes; Cobras shoot past Illinois Central, 75-69; Lady Cobras want to schore in \u2784; Koebele plays the leadhttps://spark.parkland.edu/prospectus_1984/1002/thumbnail.jp

Prospectus, April 24, 1985

https://spark.parkland.edu/prospectus_1985/1011/thumbnail.jp

Reasoning about non-functional properties using compiler intrinsic function annotations

Funding: European Union’s Horizon 2020 research and innovation programme under grant agreement No 779882.Embedded systems often need to adhere to time and energy constraints. With the increasing popularity of embed-ded systems, the interest in evaluating and optimizing non-functional properties like execution time and energy of these systems is increasing. In this paper, we describe a Resource-usage Estimate Expression Language (REEL), which allows the user to arguea bout these properties, within the source code, in a compiler understandable manner. Furthermore, we discuss the integration of REEL within a compiler framework. We, also show the propagation of REEL annotations within the compiler, and how they can be exploited to make decisions based on the non-functional properties within the source code. Finally, we explore REEL’s potential to perform ILP-based optimizations.Postprin

Prospectus, October 17, 1984

SPORTS FEATURE LADY COBRAS TAKE IV INVITATIONAL; Assistance available; many services offered; Speech team takes honors; Goal is to overthrow the \u27masculine mind\u27; PC Happenings; Parkland hosts Foley exhibit; TV for Parents and Kids debuts; Workshop focuses on sexual abuse of young children; Career restructuring focus of workshop; Hearing topic for retirees; International students enrich us all; Did You Know...; Creative Corner...Especially for you!!; Paper Dolls Souls; History; Depression; Alone; Being & Becoming; So Much; Breakable Item; Thumbism; Diary; \u27Doom Story\u27-the terror escalates; Excerpts from Some Late Evening Thoughts While Anticipating That First Great Thunderstorm of a Dry Month; Midnight; Strength or weakness; The Ultimate Weapon; Photographer captures essence of traditional, contemporary Latin America; Celebrate the Arts Week; WPCD DJ likes job, Parkland; TV programs for parents scheduled; Classifieds; Peter Pan syndrome causes relationship problems; Experience Blues Explosion; Two comedians better than one; 1978 was cruel summer for Sullivan; 1984 is proving to be a sweet fall; Drive on to educate-issues important; Powell wins invitational; Stewart places 4th; Peterson prefers to use an eight-ironhttps://spark.parkland.edu/prospectus_1984/1008/thumbnail.jp

Value Added Grains for Local and Regional Food Systems

Small grains provide numerous benefits for organic farming systems. However, because they have relatively low value and require special equipment, many organic farming systems do not integrate them. Heritage varieities of ommon wheat (Triticum aestivum ssp. Aestivum) and its antecedents spelt (Triticum aestivum, ssp. spelta), emmer (Triticum turgidum subsp. dicoccum) and einkorn (Triticum monococcum) are the subject of a project that looks at their viability. Preliminary results of variety trials show that organic farmers have several promising small grain options. Improved management and appropriate equipment are barriers to adoption that the project aims to overcome. Viability will depend on overcoming marketing as well as production obstacles