Intracellular protein delivery using QRPL - A vacuolar targeting signal on carboxypeptidase Y

Dong-Jun Park; Ngoc-Tu Nguyen; Bit-Na Kim; Yang-Hoon Kim; Jiho Min

doi:10.1016/j.enzmictec.2021.109848

Intracellular protein delivery using QRPL - A vacuolar targeting signal on carboxypeptidase Y

Enzyme Microb Technol. 2021 Sep:149:109848. doi: 10.1016/j.enzmictec.2021.109848. Epub 2021 Jun 11.

Authors

Dong-Jun Park¹, Ngoc-Tu Nguyen², Bit-Na Kim³, Yang-Hoon Kim⁴, Jiho Min⁵

Affiliations

¹ Department of Bioprocess Engineering, Jeonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, 54896, South Korea.
² Department of Bioprocess Engineering, Jeonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, 54896, South Korea; Center for Ecology and Environmental Toxicology (CEET), Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea.
³ School of Chemical Engineering, Jeonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, 54896, South Korea.
⁴ Center for Ecology and Environmental Toxicology (CEET), Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea; School of Biological Sciences, Chungbuk National University, 1 Chungdae-Ro, Seowon-Gu, Cheongju, 28644, South Korea. Electronic address: kyh@chungbuk.ac.kr.
⁵ Department of Bioprocess Engineering, Jeonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, 54896, South Korea; School of Chemical Engineering, Jeonbuk National University, 567 Baekje-daero, Deokjin-Gu Jeonju, 54896, South Korea. Electronic address: jihomin@jbnu.ac.kr.

PMID: 34311885
DOI: 10.1016/j.enzmictec.2021.109848

Abstract

The signal peptide sequence is known to increase transport efficiency to organelles in eukaryotic cells. In this study, we focus on the signal peptide of the vacuolar protein for vacuolar targeting. The signal peptide sequence QRPL of carboxypeptidase Y (CPY) was inserted inside the interest protein that does not locate in the vacuole for vacuolar targeting. We constructed recombinant strains MBTL-Q-DJ1 and MBTL-Q-DJ2 containing QRPL and green florescent protein (GFP) or aldehyde dehydrogenase 6 (ALD6), respectively. The protein location was then confirmed by confocal microscopy. Fascinatingly, the green fluorescent protein that contains QRPL inside the sequence could be expressed faster than its natural form (within 1 h after induction). Also, the aldehyde removal activity of ALD6 protein in the recombinant yeast was then analyzed by measuring the luminescent intensity in Vibrio fischeri. We confirmed that MBTL-Q-DJ2 containing ALD6 protein has the aldehydes-reducing ability, and in particular, the highest efficiency showed at 500 μg/μL of vacuolar enzyme. In summary, the signal peptide QRPL could be used not only to transport proteins accurately to vacuole but also to improve the protein activity and shorten the induction time.

Keywords: Aldehyde dehydrogenase; Formaldehyde oxidation; Lysosome-targeted drug delivery system; Signal sequence; Transfer vector; Vacuolar targeting.

MeSH terms

Cathepsin A / genetics
Protein Sorting Signals / genetics
Saccharomyces cerevisiae / genetics
Saccharomyces cerevisiae Proteins* / genetics
Vacuoles*

Substances

Protein Sorting Signals
Saccharomyces cerevisiae Proteins
Cathepsin A