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In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6\u00b0C to 64.4\u00b0C and increased its enzymatic half-life at 60\u00b0C from 5.4\u2005min to 21.9\u2005min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure\/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.<\/jats:p>","DOI":"10.1042\/bcj20190874","type":"journal-article","created":{"date-parts":[[2020,1,20]],"date-time":"2020-01-20T07:39:55Z","timestamp":1579505995000},"page":"727-745","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Lysosomal protein thermal stability does not correlate with cellular half-life: global observations and a case study of tripeptidyl-peptidase 1"],"prefix":"10.1042","volume":"477","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2480-2417","authenticated-orcid":false,"given":"Aaron M.","family":"Collier","sequence":"first","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuliya","family":"Nemtsova","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Narendra","family":"Kuber","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Whitney","family":"Banach-Petrosky","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anurag","family":"Modak","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5159-9928","authenticated-orcid":false,"given":"David E.","family":"Sleat","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."},{"name":"Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers Biomedical and Health Sciences, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2786-8347","authenticated-orcid":false,"given":"Vikas","family":"Nanda","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."},{"name":"Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers Biomedical and Health Sciences, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3148-2471","authenticated-orcid":false,"given":"Peter","family":"Lobel","sequence":"additional","affiliation":[{"name":"Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ 08854, U.S.A."},{"name":"Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers Biomedical and Health Sciences, Rutgers University, Piscataway, NJ 08854, U.S.A."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"288","published-online":{"date-parts":[[2020,2,14]]},"reference":[{"key":"2021112218054272600_BCJ-2019-0874C1","first-page":"444","volume-title":"The Neuronal Ceroid Lipofuscinoses (Batten Disease)","year":"2011","edition":"2nd ed"},{"key":"2021112218054272600_BCJ-2019-0874C2","doi-asserted-by":"publisher","first-page":"284","DOI":"10.1016\/j.gene.2016.08.031","article-title":"Analysis of large-scale whole exome sequencing data to determine the prevalence of genetically-distinct forms of neuronal ceroid lipofuscinosis","volume":"593","year":"2016","journal-title":"Gene"},{"key":"2021112218054272600_BCJ-2019-0874C3","doi-asserted-by":"publisher","first-page":"1802","DOI":"10.1126\/science.277.5333.1802","article-title":"Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis","volume":"277","year":"1997","journal-title":"Science"},{"key":"2021112218054272600_BCJ-2019-0874C4","doi-asserted-by":"publisher","first-page":"496","DOI":"10.1016\/S0167-4838(98)00238-6","article-title":"Tripeptidyl-peptidase I is apparently the CLN2 protein absent in classical late-infantile neuronal ceroid lipofuscinosis","volume":"1429","year":"1999","journal-title":"Biochim. 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