Over-expression of a Human CD62L Ecto-domain and a Potential Role of RNA Pseudoknot Structures in Recombinant Protein Expression

Overview

Journal Protein Expr Purif

Specialty Molecular Biology

Date 2017 Aug 27

PMID 28842197

Citations 1

Authors

Matthew Spencer

Nathan Max

Joanna Ireland

Zhongcheng Zou

Ruipeng Wang

Peter Sun

Affiliations

Soon will be listed here.

Abstract

L-selectin (CD62L) is an extracellular protein with a lectin-like domain that mediates rolling adhesion of leukocytes to vascular endothelial cell surfaces. Currently, there are no solved structures for the ectodomain of CD62L, nor of CD62L in complex with its ligand. We have developed a rapid mammalian recombinant protein expression system using an amplifiable glutamine synthase based vector. Here, we further developed and applied this method to express and purify the entire extracellular region of CD62L. This resulted in excess of 20 mg/L yield of recombinant CD62L. In an attempt to understand the different expression levels among four similar CD62L constructs that differ primarily in signal sequences, we calculated the presence of potential RNA pseudoknots in their signal sequences. The results showed the presence of pseudoknots involving the start codon and between the signal sequence and gene in the mRNA of the non-expressing constructs, suggesting a potential inhibitory role of RNA pseudoknots in recombinant protein expression.

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References

Cash D, Cohen-Zontag O, Kim N, Shefer K, Brown Y, Ulyanov N . Pyrimidine motif triple helix in the Kluyveromyces lactis telomerase RNA pseudoknot is essential for function in vivo. Proc Natl Acad Sci U S A. 2013; 110(27):10970-5. PMC: 3704002. DOI: 10.1073/pnas.1309590110. View

Gifford J, Ishida H, Vogel H . Structural insights into calmodulin-regulated L-selectin ectodomain shedding. J Biol Chem. 2012; 287(32):26513-27. PMC: 3410993. DOI: 10.1074/jbc.M112.373373. View

Sperschneider J, Datta A, Wise M . Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins. RNA. 2010; 17(1):27-38. PMC: 3004063. DOI: 10.1261/rna.2394511. View

Peselis A, Serganov A . Structure and function of pseudoknots involved in gene expression control. Wiley Interdiscip Rev RNA. 2014; 5(6):803-22. PMC: 4664075. DOI: 10.1002/wrna.1247. View

Giedroc D, Theimer C, Nixon P . Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting. J Mol Biol. 2000; 298(2):167-85. PMC: 7126452. DOI: 10.1006/jmbi.2000.3668. View

Zou Z, Sun P . An improved recombinant mammalian cell expression system for human transforming growth factor-beta2 and -beta3 preparations. Protein Expr Purif. 2006; 50(1):9-17. DOI: 10.1016/j.pep.2006.06.022. View

Wigler M, Perucho M, Kurtz D, Dana S, Pellicer A, Axel R . Transformation of mammalian cells with an amplifiable dominant-acting gene. Proc Natl Acad Sci U S A. 1980; 77(6):3567-70. PMC: 349658. DOI: 10.1073/pnas.77.6.3567. View

Somers W, Tang J, Shaw G, Camphausen R . Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1. Cell. 2000; 103(3):467-79. DOI: 10.1016/s0092-8674(00)00138-0. View

Patnaik S, Stanley P . Lectin-resistant CHO glycosylation mutants. Methods Enzymol. 2006; 416:159-82. DOI: 10.1016/S0076-6879(06)16011-5. View

10.

Holland J, Hansen M, Du Z, Hoffman D . An examination of coaxial stacking of helical stems in a pseudoknot motif: the gene 32 messenger RNA pseudoknot of bacteriophage T2. RNA. 1999; 5(2):257-71. PMC: 1369757. DOI: 10.1017/s1355838299981360. View

11.

Staple D, Butcher S . Pseudoknots: RNA structures with diverse functions. PLoS Biol. 2005; 3(6):e213. PMC: 1149493. DOI: 10.1371/journal.pbio.0030213. View

12.

Ley K . The role of selectins in inflammation and disease. Trends Mol Med. 2003; 9(6):263-8. DOI: 10.1016/s1471-4914(03)00071-6. View

13.

Plant E, Muldoon Jacobs K, Harger J, Meskauskas A, Jacobs J, Baxter J . The 9-A solution: how mRNA pseudoknots promote efficient programmed -1 ribosomal frameshifting. RNA. 2003; 9(2):168-74. PMC: 1237042. DOI: 10.1261/rna.2132503. View

14.

Kozak M . Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs. Mol Cell Biol. 1989; 9(11):5134-42. PMC: 363665. DOI: 10.1128/mcb.9.11.5134-5142.1989. View

15.

Pettersen E, Goddard T, Huang C, Couch G, Greenblatt D, Meng E . UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13):1605-12. DOI: 10.1002/jcc.20084. View

16.

Pleij C, Rietveld K, Bosch L . A new principle of RNA folding based on pseudoknotting. Nucleic Acids Res. 1985; 13(5):1717-31. PMC: 341107. DOI: 10.1093/nar/13.5.1717. View

17.

Brown H, Roth G, Holzapfel G, Shen S, Rahbari K, Ireland J . Development of an improved mammalian overexpression method for human CD62L. Protein Expr Purif. 2014; 105:8-13. PMC: 4385751. DOI: 10.1016/j.pep.2014.09.018. View

18.

Schimke R . Methotrexate resistance and gene amplification. Mechanisms and implications. Cancer. 1986; 57(10):1912-7. DOI: 10.1002/1097-0142(19860515)57:10<1912::aid-cncr2820571004>3.0.co;2-o. View

19.

Sperschneider J, Datta A . DotKnot: pseudoknot prediction using the probability dot plot under a refined energy model. Nucleic Acids Res. 2010; 38(7):e103. PMC: 2853144. DOI: 10.1093/nar/gkq021. View

20.

Zou Z, Sun P . Overexpression of human transforming growth factor-beta1 using a recombinant CHO cell expression system. Protein Expr Purif. 2004; 37(2):265-72. DOI: 10.1016/j.pep.2003.06.001. View