LARGE-SCALE PURIFICATION AND REFOLDING OF HIV-1 PROTEASE FROM ESCHERICHIA-COLI INCLUSION-BODIES

被引：43

作者：

HUI, JO ^{[1
]}

TOMASSELLI, AG ^{[1
]}

REARDON, IM ^{[1
]}

LULL, JM ^{[1
]}

BRUNNER, DP ^{[1
]}

TOMICH, CSC ^{[1
]}

HEINRIKSON, RL ^{[1
]}

机构：

[1] UPJOHN CO,301 HENRIETTA ST,KALAMAZOO,MI 49001

来源：

JOURNAL OF PROTEIN CHEMISTRY | 1993年 / 12卷 / 03期

关键词：

HIV-1; PROTEASE; GEL-FILTRATION; SUPERDEX-75; FPLC COLUMN; REVERSED-PHASE HPLC;

D O I：

10.1007/BF01028194

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) almost-equal-to 1 0,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

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页码：323 / 327

页数：5

相关论文

共 15 条

[1] AN INHIBITOR OF THE PROTEASE BLOCKS MATURATION OF HUMAN AND SIMIAN IMMUNODEFICIENCY VIRUSES AND SPREAD OF INFECTION [J].

ASHORN, P ;

MCQUADE, TJ ;

THAISRIVONGS, S ;

TOMASSELLI, AG ;

TARPLEY, WG ;

MOSS, B .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (19) :7472-7476

[2] GENETIC-LOCUS, PRIMARY STRUCTURE, AND CHEMICAL SYNTHESIS OF HUMAN IMMUNODEFICIENCY VIRUS PROTEASE [J].

COPELAND, TD ;

OROSZLAN, S .

GENE ANALYSIS TECHNIQUES, 1988, 5 (06) :109-115

[3]

IDO E, 1991, J BIOL CHEM, V266, P24359

[4] ACTIVE HUMAN IMMUNODEFICIENCY VIRUS PROTEASE IS REQUIRED FOR VIRAL INFECTIVITY [J].

KOHL, NE ;

EMINI, EA ;

SCHLEIF, WA ;

DAVIS, LJ ;

HEIMBACH, JC ;

DIXON, RAF ;

SCOLNICK, EM ;

SIGAL, IS .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (13) :4686-4690

[5] CLEAVAGE OF STRUCTURAL PROTEINS DURING ASSEMBLY OF HEAD OF BACTERIOPHAGE-T4 [J].

LAEMMLI, UK .

NATURE, 1970, 227 (5259) :680-+

[6]

LEATHERBARROW RJ, 1988, ENZFITTER

[7] CHEMICAL SYNTHESIS AND ENZYMATIC-ACTIVITY OF A 99-RESIDUE PEPTIDE WITH A SEQUENCE PROPOSED FOR THE HUMAN IMMUNODEFICIENCY VIRUS PROTEASE [J].

NUTT, RF ;

BRADY, SF ;

DARKE, PL ;

CICCARONE, TM ;

COLTON, CD ;

NUTT, EM ;

RODKEY, JA ;

BENNETT, CD ;

WAXMAN, LH ;

SIGAL, IS ;

ANDERSON, PS ;

VEBER, DF .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1988, 85 (19) :7129-7133

[8] RECOMBINANT HIV1 PROTEASE SECRETED BY SACCHAROMYCES-CEREVISIAE CORRECTLY PROCESSES MYRISTYLATED GAG POLYPROTEIN [J].

PICHUANTES, S ;

BABE, LM ;

BARR, PJ ;

CRAIK, CS .

PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1989, 6 (03) :324-337

[9] ENZYMATIC-ACTIVITY OF A SYNTHETIC-99 RESIDUE PROTEIN CORRESPONDING TO THE PUTATIVE HIV-1 PROTEASE [J].

SCHNEIDER, J ;

KENT, SBH .

CELL, 1988, 54 (03) :363-368

[10] CHARACTERIZATION AND AUTOPROCESSING OF PRECURSOR AND MATURE FORMS OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 (HIV-1) PROTEASE PURIFIED FROM ESCHERICHIA-COLI [J].

STRICKLER, JE ;

GORNIAK, J ;

DAYTON, B ;

MEEK, T ;

MOORE, M ;

MAGAARD, V ;

MALINOWSKI, J ;

DEBOUCK, C .

PROTEINS-STRUCTURE FUNCTION AND GENETICS, 1989, 6 (02) :139-154