An open library of human kinase domain constructs for automated bacterial expression

Preprint OPEN
Rodríguez-Laureano, Lucelenie ; Işık, Mehtap ; Chodera, John ; Seeliger, Markus ; Jeans, Chris ; Gradia, Scott ; Hanson, Sonya ; Parton, Daniel ; Albanese, Steven ; Levinson, Nicholas ; Behr, Julie (2017)
  • Related identifiers: doi: 10.1101/038711
  • Subject: bepress|Life Sciences|Biology | bepress|Life Sciences|Biochemistry, Biophysics, and Structural Biology|Biochemistry

Kinases play a critical role in many cellular signaling pathways and are dysregulated in a number of diseases, such as cancer, diabetes, and neurodegeneration. Since the FDA approval of imatinib in 2001, therapeutics targeting kinases now account for roughly 50% of current cancer drug discovery efforts. The ability to explore human kinase biochemistry, biophysics, and structural biology in the laboratory is essential to making rapid progress in understanding kinase regulation, designing selective inhibitors, and studying the emergence of drug resistance. While insect and mammalian expression systems are frequently used for the expression of human kinases, bacterial expression systems are superior in terms of simplicity and cost-effectiveness but have historically struggled with human kinase expression. Following the discovery that phosphatase coexpression could produce high yields of Src and Abl kinase domains in bacterial expression systems, we have generated a library of 52 His-tagged human kinase domain constructs that express above 2 ug/mL culture in a simple automated bacterial expression system utilizing phosphatase coexpression (YopH for Tyr kinases, Lambda for Ser/Thr kinases). Here, we report a structural bioinformatics approach to identify kinase domain constructs likely to express in bacteria, experiments demonstrating our simple construct selection strategy selects constructs with good expression yields in a test of 84 potential kinase domain boundaries for Abl, and yields from a high-throughput expression screen of 96 human kinase constructs. We also demonstrate how the resulting expressing constructs can be used for the biophysical and biochemical study of clinical mutations by engineering a panel of 48 Src mutations and 46 Abl mutations via single-primer mutagenesis and screening the resulting library for expression yields. The wild-type kinase construct library is available publicly via addgene, and should prove to be of high utility for experiments focused on drug discovery and the emergence of drug resistance.
  • References (29)
    29 references, page 1 of 3

    Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002; 298:1912-1934.

    Médard G, Pachl F, Ruprecht B, Klaeger S, Heinzlmeir S, Helm D, Qiao H, Ku X, Wilhelm M, Kuehne T, Wu Z, Dittmann A, Hopf C, Kramer K, Kuster B. Optimized chemical proteomics assay for kinase inhibitor profiling. Journal of proteome research. 2015 Mar; 14(3):1574-1586.

    477 Nagar B, Hantschel O, Young MA, Scheffzek K, Veach D, Bornmann W, Clarkson B, Superti-Furga G, Kuriyan J.

    478 Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell. 2003 Mar; 112(6):859-871.

    479 Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MG Maureen F nd Kris, Varmus H. Acquired resistance 480 of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase 481 domain. PLoS medicine. 2005 Mar; 2(3):e73.

    482 Politi K, Ayeni D, Lynch T. The Next Wave of EGFR Tyrosine Kinase Inhibitors Enter the Clinic. Cancer Cell. 2015 483 Jun; 27(6):751-753.

    484 Ramos AH, Lichtenstein L, Gupta M, Lawrence MS, Pugh TJ, Saksena G, Meyerson M, Getz G. Oncotator: cancer 485 variant annotation tool. Human mutation. 2015 Apr; 36(4):E2423-9.

    486 Reva B, Antipin Y, Sander C. Determinants of protein function revealed by combinatorial entropy optimiza487 tion. . 2007; 8(11):R232., doi: 488 10.1186/gb-2007-8-11-r232.

    489 Reva B, Antipin Y, Sander C. Predicting the functional impact of protein mutations: application to can490 cer genomics. Nucleic Acids Research. 2011; 39(17):e118., doi: 491 10.1093/nar/gkr407.

    492 Roskoski Jr R, USFDA approved protein kinase inhibitors; 2017., updated 3 493 May 2017.

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