Optimizing HIV-1 protease production in Escherichia coli as fusion protein

被引:28
作者
Volonte, Federica [1 ,2 ,3 ]
Piubelli, Luciano [1 ,2 ,3 ]
Pollegioni, Loredano [1 ,2 ,3 ]
机构
[1] Univ Insubria, Dipartimento Biotecnol & Sci Mol, I-21100 Varese, Italy
[2] Politecn Milan, Ctr Interuniv Ric Biotecnol Prot, I-20131 Milan, Italy
[3] Univ Insubria, I-20131 Milan, Italy
来源
MICROBIAL CELL FACTORIES | 2011年 / 10卷
关键词
CHOLESTEROL OXIDASE; INCLUSION-BODIES; EXPRESSION; PURIFICATION; CLONING;
D O I
10.1186/1475-2859-10-53
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background: Human immunodeficiency virus (HIV) is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr) is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results: A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr) was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA) or glutathione S-transferase (GST), also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis) and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3)-RIL host and in TB or M9 medium to which 1% (w/v) glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts) and a growth temperature of 37 C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth). GST:HIVPr was in part (50%) produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity <= 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded similar to 0.15 mg of pure HIV-1Pr per liter. Conclusions: By using this optimized expression and purification procedure fairly large amounts of good-quality HIV-1Pr recombinant enzyme can be produced at the lab-scale and thus used for further biochemical studies.
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页数:10
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共 25 条
  • [1] A folding inhibitor of the HIV-1 protease
    Broglia, RA
    Provasi, D
    Vasile, F
    Ottolina, G
    Longhi, R
    Tiana, G
    [J]. PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS, 2006, 62 (04) : 928 - 933
  • [2] Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin
    Caldinelli, L
    Iametti, S
    Barbiroli, A
    Bonomi, F
    Fessas, D
    Molla, G
    Pilone, MS
    Pollegioni, L
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2005, 280 (24) : 22572 - 22581
  • [3] HIGH-LEVEL SYNTHESIS OF RECOMBINANT HIV-1 PROTEASE AND THE RECOVERY OF ACTIVE ENZYME FROM INCLUSION-BODIES
    CHENG, YSE
    MCGOWAN, MH
    KETTNER, CA
    SCHLOSS, JV
    ERICKSONVIITANEN, S
    YIN, FH
    [J]. GENE, 1990, 87 (02) : 243 - 248
  • [4] HIV-I protease - Cloning, expression, and purification
    Dergousova, NI
    Amerik, AY
    Volynskaya, AM
    Rumsh, LD
    [J]. APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, 1996, 61 (1-2) : 97 - 107
  • [5] A REGULATORY CASCADE IN THE INDUCTION OF RHABAD
    EGAN, SM
    SCHLEIF, RF
    [J]. JOURNAL OF MOLECULAR BIOLOGY, 1993, 234 (01) : 87 - 98
  • [6] Fantinato S., 2001, ENZYME MICROB TECHNO, V29, P407
  • [7] LARGE-SCALE PRODUCTION OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI USING SELECTIVE EXTRACTION AND MEMBRANE FRACTIONATION
    GUSTAFSON, ME
    JUNGER, KD
    FOY, BA
    BAEZ, JA
    BISHOP, BF
    RANGWALA, SH
    MICHENER, ML
    LEIMGRUBER, RM
    HOUSEMAN, KA
    MUELLER, RA
    MATTHEWS, BK
    OLINS, PO
    GRABNER, RW
    HERSHMAN, A
    [J]. PROTEIN EXPRESSION AND PURIFICATION, 1995, 6 (04) : 512 - 518
  • [8] Use of new methods for construction of tightly regulated arabinose and rhamnose promoter fusions in studies of the Escherichia coli phosphate regulon
    Haldimann, A
    Daniels, LL
    Wanner, BL
    [J]. JOURNAL OF BACTERIOLOGY, 1998, 180 (05) : 1277 - 1286
  • [9] PARTIAL-PURIFICATION AND SUBSTRATE ANALYSIS OF BACTERIALLY EXPRESSED HIV PROTEASE BY MEANS OF MONOCLONAL-ANTIBODY
    HANSEN, J
    BILLICH, S
    SCHULZE, T
    SUKROW, S
    MOELLING, K
    [J]. EMBO JOURNAL, 1988, 7 (06) : 1785 - 1791
  • [10] The packaging and maturation of the HIV-1 pol proteins
    Hill, M
    Tachedjian, G
    Mak, J
    [J]. CURRENT HIV RESEARCH, 2005, 3 (01) : 73 - 85