Rapid and simple purification of human immunodeficiency virus 1 epitope gp41

Journal of Virological Methods, 41 (1993) 93-100 0 1993 Elsevier Science Publishers B.V. / All rights

VIRMET

93

reserved / 0166-0934/93/$05.00

01430

Rapid and simple purification of human immunodefkiency virus 1 epitope gp41 Mi Jin Sohn, Seo Hee Cho, Won-Hee Jang, Young-Hae Sang-Uk Nham and Young-Ik Lee Laboratory

Chong,

of‘ Molecular Genetics, Genetic Engineering Research Institute, Korea Institute and Technology,

of Science

Daeduk, Daejun (Korea)

(Accepted 31 July 1992)

Summary In order to develop a reliable and inexpensive serodiagnostic method, part of the transmembrane glycoprotein gene of HIV-l, gp41’, (HIV-env 548-646) was cloned into an expression vector, pCTl0 with a sequence encoding a hydroxylamine cleavage site and with a part of Lac Z gene (Lac 2”: 834 base pairs) as a fusion partner. Overexpression of Lac Z”-gp41’ was induced in E. co/i and the gp41’ fusion protein was purified to homogeneity by centrifugation, hydroxylamine cleavage and an ion-exchange chromatography. Western blot analysis and enzyme-linked immunosorbant assay (ELISA) using the purified gp41 fragment showed high sensitivity and specificity of gp41 as an antigen to detect anti HIV-l antibodies in testing human sera. These results suggest that this simple and rapid purification method is reliable for obtaining a large quantity of purified gp41’. Human

immunodeficiency

virus-l;

gp41; Purification

Introduction Human immunodeficiency virus type 1 (HIV-l) is a causative agent of Acquired Immune Deficiency Syndrome (AIDS) (Fauci, 1988). The coat of HIV-I consists of ey1vgene products which elicit an immune response in most HIV-l infected individuals. The env gene of this virus encodes a precursor Correspondence Institute, Korea

to: Y.-I. Lee, Laboratory of Molecular Genetics, Institute of Science and Technology, Daeduk, Daejun,

Genetic 3055333,

Engineering Korea.

Research,

94

peptide (gpl60) which is processed later to the external envelope glycoprotein (gpl20) and transmembrane protein (gp41) (Veronese et al., 1985). While there are highly variable regions in the viral env gene products, the C terminal portion of gpl20 and N terminal region of gp41 are known to be less variable and antigenic (Starcich et al., 1986). These proteins, therefore, could be valuable as antigens for detecting HIV-l antibody. Several diagnostic methods have been developed, which detect antibody against HIV-l antigen in human blood. Heterologous expression of HIV-l genes such as gag or env in E. coli have been employed for the development of reliable and inexpensive diagnostic reagents (Crow1 et al., 1985; Windheuser et al., 1988). One problem with the current diagnostic procedures which use bacterially produced proteins is that the presence of bacterial protein contaminants in the purified HIV-l antigens often causes false positive reactions. A number of methods to express and to purify HIV-l antigens have been reported in an effort to eliminate these problems (Chang et al., 1985; Ellinger et al., 1991). A simple and rapid purification method for a HIV-1 antigen is described in an attempt to establish reliable and inexpensive serodiagnostic procedure. A part of gp41 (gp41’: HIV-env a.a. 548-646) was overexpressed in E. co/i and purified to homogeneity in a large quantity.

Materials and Methods Plasmids A plasmid, pSP64-BHlO (Ratner et al., 1985), containing HIV-l gene was obtained from R. Gallo (NIH, USA). The expression vector pCTl0 contains tat promoter, Lac Z (1062 bp) and lac repressor. The expression plasmid ‘cassette’ pATHJ described previously (Yoo et al., 1991), contains trp promoter, trp E, AACGGC sequence encoding hydroxylamine cleavage site (Asp-Pro) and a multicloning site. Construction

of’ a gp41’ expression plasmid

Expression vector pYCM-env2 was constructed by standard procedures (Sambrook et al., 1989). Briefly, pSP64-BHlO was digested by HaeIII and Hind111 to yield a 297 bp DNA fragment which is in the region of N-terminal portion of HIV-l transmembrane protein, gp41 (HIV-env a.a. 548-646). This fragment was ligated into an expression cassette pATHJ, and a 110 bp MspI and C/a1 fragment containing a hydroxylamine cleavage site and gp41’ was isolated from the constructed plasmid. This fragment was then cloned into a ClaI site of pCTl0 to yield an expression plasmid, pYCM-env2 with tat promoter and a bacterial fusion gene Lac Z” (a part of Lac Z: 834 bp). A stop codon was provided by filling in reaction after ClaI digestion.

95

Expression and purification

of gp41’

Bacterial strain JM109 containing pYCM-env2 was grown overnight in LB medium containing ampicillin at a concentration of 20 pg/ml. Five ml of overnight culture was inoculated into 200 ml LB medium and mixed with fresh 4 liter LB medium after 2-3 h incubation. Isopropyl-1-thio-beta-D-galactoside (IPTG) (1 mM) was added to the culture when the cell had reached early log phase (O.D. 600 = 0.4). Cells were harvested after 6-7 h incubation at 37°C. The inducibility of the fused gene was determined by sodium dodecyl sulfatepoly acrylamide gel electrophoresis (SDS-PAGE). Most of the induced gp41’ fusion protein existed in inclusion bodies and was easily collected by centrifugation at 5000 x g after sonication, or passing them through French presser cells (Aminco). Pellets containing inclusion bodies were washed with 3 M urea in 50 mM Tris-HCl (pH 7.0) to reduce non-specific association of bacterial proteins. The inclusion body pellets, then, solubilized in 30 ml of Buffer A (5.0 M urea, 50 mM Tris-HCl pH 7.0, 10 mM dithiothreitol, 1.0 mM EDTA). To this solution, hydroxylamine was added to 2.0 M final concentration. The cleavage reaction was carried out at 45°C for 4 h with constant shaking and was subsequently terminated by the addition of concentrated formic acid to pH 4.0. The solution was then placed in a dialysis membrane and dialysed against distilled water. gp41’ precipitated during dialysis and was collected by a simple centrifugation. The pellets dissolved in Buffer A was loaded onto a DEAE-Sepharose column (3.0 cm x 10 cm) previously equilibrated with buffer A and then eluted with the same buffer. Each fraction was subjected to SDS-PAGE to determine the presence of gp41’. Enzyme-linked

immunosorbent

assay (ELISA)

For ELISA, wells of polyvinyl microtiter plates were coated with gp41’(10 pg/ml) solubilized in 5 M Urea in phosphate-buffered saline (PBS) overnight, then blocked with 5% skim milk in PBS for 2 h. HIV-l positive sera (obtained from National Institute of Health, KOREA) or normal sera from healthy donors were serially diluted with 50 ~1 of the blocking buffer in each well and incubated for 2 h. After three washes with PBS, 50 ~1 of horseradish peroxidase conjugated goat antihuman IgG were added to each well (2 h at 37°C). The plates were then washed with PBS to remove unbound conjugate and 100 ~1 of 0.2 mM ABTS [2,2’-azino-di(3-ethylbenzothiazoline sulfonate)] and H202 solution was added. The calorimetric reaction was terminated by the addition of 25 ~1 of 2 N NaOH and the color intensity was measured at 405 nm with an automatic microtiter reader.

96

Results and Discussion

Construction and expression ofgp41’ ~ont~in~~gvector The amino terminal portion of gp41 is an external region of transmembrane protein which binds to the gpl20 of HIV-I with hydrophobic interaction (Kowalski et al., 1987). Since this region of gp41 is highly conserved (Starcich et al., 1986) and is strongly antigenic, this fragment of gp41 is known to be useful to diagnose HIV-l infection (Gnann et al., 1987; Klasse et al., 1988). To obtain a large quantity of gp41, 297 bp DNA from the N-terminus of gp41 gene (encoding HIV-env amino acids 548-646) was cloned into a pCTl0 plasmid with a hydroxylamine cleavage site and Lac Z”. Fig. 1 shows the structure of the resulting expression plasmid for gp41’ production, pYCMenv2. The vector pCTl0 was chosen because: (1) it provided a strong promoter (Ptac) and (2) exhibits an appropriate size for a fusion partner, Lac Z” (a part of Lac 2;: 834 bp) after Cl& digestion (Choi et al., 1988). To eliminate fused Lac Z” gene product after a high level of expression, we inserted a synthetic oligonucleotide encoding Asn-Gly linker which also provided a hydroxylamine cleavage site between Lac Z” and gp41’. A transcription terminator (T) is located at the 3’ end of Lac Z and a translational termination codon is at the end of gp41’ (Fig. 1). beta-gal--ACG

GGG AAT TCG AAC GGC GGG GAT CCC AGA-EamH HA sits ECORl

\

Fig. 1. The structure of pYCM-env2 expression encoding gp41’. A hydroxyiamine cleavage site genes, and NindIIl site and a termjnation codon gp41’ gene due to these ancillary sequences. A

plasmid. 0.8 kb Lac Z” (beta-gal) was fused with a gene as well as EcoRI and BarnHI sites are in between the two locate at the 3’ of gp41’ gene. 45 nucleotides were added to transcription terminator (T) was located at the 3’ end of Lac 2.

91

Expression and pur$ication of gp41’ The fused gene was overexpressed in E. coli following induction by 1.0 mM IPTG. The production of the fusion gene product reached as high as 40% of total cellular proteins as shown in Fig. 2 (lane 2). The length of time of induction for the plasmid appears to be critical to get high level expression. Short induction times caused low levels of expression, whereas long induction times, such as overnight, introduced outgrowth of non-plasmid bearing bacteria, leading to a low yield of the protein of interest. A 6-7 h incubation was optimal to reach maximal levels of gp41’ expression. We determined that most of the fusion proteins were in the form of inclusion bodies. Washing the inclusion bodies with 3 M urea provided additional purity by removing associated bacterial cellular proteins. (Fig. 2, lane 4). To remove Lac Z” product from gp41’, hydroxylamine cleavage was carried out. Employing a chemical cleavage was the method of choice in this study since there is no hydroxylamine site in gp41’ and inclusion bodies had to be

gP41

Fig. 2. SDS-PAGE of HIV-I en” gene products. Various samples prepared by the method described in Materials and Methods were subjected to 15% polyacrylamide gel electrophoresis and stained by Coomassie blue. Lane 1: uninduced JM 109 cells; lane 2: induced by 1.O mM IPTG; lane 3: inclusion bodies solubilized in Buffer A; lane 4: 5 M urea-washed inclusion bodies; lane 5: hydroxylamine-cleaved fusion protein; lane 6: purified gp41’.

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solubilized in high concentration of urea or guanidine chloride in which most enzymes are in fact not able to work. The efficiency of this reaction was generally 6&80% as shown in SDS-PAGE (Fig. 2, lane 5). Proteins subjected to hydroxylamine cleavage were dialysed against distilled water overnight. Because of low solubility of gp41’, it precipitated during dialysis step. This property provided a convenient way of handling a large quantity of the protein. Pellets collected by a simple centrifugation were solubilized in a buffer containing 5 M urea (namely buffer A) and subjected further to an ionexchange column chromatography. As shown in Fig. 3, gp41’ was separated successfully from other proteins in the first unbound peak. SDS-PAGE analysis shows the homogeneity of the purified gp41’ (Figs. 3 and 2, lane 6). Most nonspecific bacterial proteins were bound to the DEAE resin in buffer A while the gp41’ eluted as an unbound fraction. The purification procedure described here is very simple and rapid, and the average yield of gp41’ is about 10-15 mg/l of culture medium. Therefore, this method would be very useful for the production of gp41’ on a large scale.

FRACTION

NUMBER

Fig. 3. Elution profile of gp41’ from a DEAE-Sepharose column. Hydroxylamine cleaved gene products were precipitated and then dissolved in buffer A. After dialysis against buffer A, protein samples were eluted from a DEAE-Sepharose column. The 0.1 ml samples from each tubes were analyzed to determine the presence of gp41’. 1: first unbound peak; 2: the second unbound peak; 3: eluted proteins by 0.5 mM NaCl in buffer A.

99

TABLE I Results of testing 36 antibody-positive

and 266 antibody-negative

plasma

Specimen tested

“No. positive/total

Sensitivity

HIV-I + plasma based on immunoblot HIV-I- plasma based on immunoblot

36136 l/266

100%

Specificity 99.6%

“Evaluation as positive or negative is based on an OD 405of greater or less than the established cutoff OD4s5 = 0.250 for each ELISA assay, respectively.

ELISA In order to determine the specificity and sensitivity of gp41’ as an antigen for detecting antibodies in HIV-l positive human sera, a quantitive immunoassay was carried out. Purified gp41’ (1 pg/well) was coated on the wells of polyvinyl microtiter plates. 5% skimmed milk were used as a blocking agent, and 36 HIV-l positive plasma (obtained from Korea Institute of Health) as well as 266 plasma from healthy donors were used as testing samples. The optimal screening dilution factor was the range 400 to 12 800 and the end point titer is higher than 25 600 in this analysis (data not shown). Purified gp41’ reacted only to antibodies in the HIV-l positive plasma except one false positive case, showing a high sensitivity (36/36, 100%) and a specificity (265/266, 99.6%) (Table 1). This result also reflects the reliability of the purification procedure to eliminate bacterial contaminants from gp41’, because most false positive reactions are derived from contaminated bacterial proteins which are often recognized by the antibodies in testing human sera. Taken together, these data show that the purified gp41’ by the simple method described here can be used as an antigen for diagnostic reagents. The method reported in this study using Lac Z gene and tat promoter could be applied for the production of several viral antigens in bacteria. These viral antigens could offer reliable diagnostic reagents with specificity and sensitivity.

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