TY - JOUR
T1 - Purification and characterization of human immunodeficiency virus type 1 reverse transcriptase
AU - Le Grice, Stuart F.J.
AU - Cameron, Craig E.
AU - Benkovic, Stephen J.
N1 - Funding Information:
S. F. J. Le Grice is supported by grants AI 31147 and GM 46623 from the National Institutes of Health. S. J. Benkovici s supported by grant GM13306 from the National Institutes of Health, C. E. Cameron is supported by fellowship 41 09076 from the National Institutes of Health. A portion of the work presented here was also supported by a NATO collaborative research grant (CRG 900.471) to S. F. J. Le Grice. The technical assistance of K. J. Howard in preparation of p66/p51 HIV-1 RT variants is gratefully acknowledged.
Funding Information:
We thank B6n6dicte Purcell and Stephen Oliver for the plasmid YCR14C. This work was supported by grants from the USPHS (GM25508 and GM47281) and California TRDRP 3RT-0190.
PY - 1995/1/1
Y1 - 1995/1/1
N2 - This chapter discusses the purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Metal chelate chromatography is now finding widespread use in the purification of human immunodeficiency virus (HIV) reverse transcriptase (RT) directly from the high-speed supernatant of bacterial homogenates. Metal chelate affinity chromatography offers a rapid and highly reproducible means of preparing (1) the individual p66 and p51 HIV RT subunits, (2) heterodimer p66/p51, and (3) reconstituted, selectively modified heterodimer directly from bacterial homogenates. The application of a highly selective affinity matrix as the primary purification step has the advantage that bacterial proteases are eliminated at an early stage, avoiding proteolysis of the reconstituted protein. The ease with which HIV RT can be purified by metal chelate affinity chromatography has prompted to develop methodologies for protein mini preparation, where four small columns can be run simultaneously and RT eluted in a batch wise fashion. This procedure has been proven successful in cases where individual domains of the enzyme have been analyzed by insertional mutagenesis.
AB - This chapter discusses the purification and characterization of human immunodeficiency virus type 1 reverse transcriptase. Metal chelate chromatography is now finding widespread use in the purification of human immunodeficiency virus (HIV) reverse transcriptase (RT) directly from the high-speed supernatant of bacterial homogenates. Metal chelate affinity chromatography offers a rapid and highly reproducible means of preparing (1) the individual p66 and p51 HIV RT subunits, (2) heterodimer p66/p51, and (3) reconstituted, selectively modified heterodimer directly from bacterial homogenates. The application of a highly selective affinity matrix as the primary purification step has the advantage that bacterial proteases are eliminated at an early stage, avoiding proteolysis of the reconstituted protein. The ease with which HIV RT can be purified by metal chelate affinity chromatography has prompted to develop methodologies for protein mini preparation, where four small columns can be run simultaneously and RT eluted in a batch wise fashion. This procedure has been proven successful in cases where individual domains of the enzyme have been analyzed by insertional mutagenesis.
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U2 - 10.1016/0076-6879(95)62015-X
DO - 10.1016/0076-6879(95)62015-X
M3 - Article
C2 - 8594344
AN - SCOPUS:0028789990
SN - 0076-6879
VL - 262
SP - 130
EP - 144
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -