                         SEQUENCE LISTING

<110>  Rao, Venigalla B.
       Alsalmi, Wadad
 
<120>  A NEW APPROACH TO PRODUCE HIV-1 GP140 ENVELOPE PROTEIN TRIMERS

<130>  00719.42.0004

<140>  14/806735
<141>  2015-07-23

<150>  62133578
<151>  2015-03-16

<150>  62166271
<151>  2015-05-26

<160>  13    

<170>  PatentIn version 3.5

<210>  1
<211>  27
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial foldon protein sequence derived from bacteriophage T4.

<400>  1

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 
1               5                   10                  15      


Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 
            20                  25          


<210>  2
<211>  8
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artifical Strep-Tag II protein sequence

<400>  2

Trp Ser His Pro Gln Phe Glu Lys 
1               5               


<210>  3
<211>  8
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial octa-histidine tag protein sequence

<400>  3

His His His His His His His His 
1               5               


<210>  4
<211>  6
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial hexa-histidine tag protein sequence

<400>  4

His His His His His His 
1               5       


<210>  5
<211>  23
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial peptide linker protein sequence

<400>  5

Ala Ala Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly 
1               5                   10                  15      


Gly Gly Ser Gly Gly Ser Ala 
            20              


<210>  6
<211>  31
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial peptide linker protein sequence

<400>  6

Ala Ala Ala Leu Glu Val Leu Phe Gln Gly Pro Trp Ser His Pro Gln 
1               5                   10                  15      


Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala 
            20                  25                  30      


<210>  7
<211>  35
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial peptide linker protein sequence

<400>  7

Ala Ala Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Trp 
1               5                   10                  15      


Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly 
            20                  25                  30          


Gly Ser Ala 
        35  


<210>  8
<211>  43
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial peptide linker protein sequence

<400>  8

Ala Ala Ala Leu Glu Val Leu Phe Gln Gly Pro Gly Gly Gly Ser Gly 
1               5                   10                  15      


Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly 
            20                  25                  30          


Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala 
        35                  40              


<210>  9
<211>  27
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial peptide linker protein sequence

<400>  9

Ala Ala Ala Leu Glu Val Leu Phe Gln Gly Pro Ala Pro Ala Pro Ala 
1               5                   10                  15      


Pro Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 
            20                  25          


<210>  10
<211>  659
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial protein sequence of Strep-Tagged JRFL 
       SOSIP(1-5).R6.664 gp140 comprising an engineered HIV-1 clade B 
       JRFL gp140,  a peptide linker, and a Strep-Tag.

<400>  10

Val Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 
1               5                   10                  15      


Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp 
            20                  25                  30          


Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp 
        35                  40                  45              


Pro Asn Pro Gln Glu Val Val Leu Glu Asn Val Thr Glu His Phe Asn 
    50                  55                  60                  


Met Trp Lys Asn Asn Met Val Glu Gln Met Gln Glu Asp Ile Ile Ser 
65                  70                  75                  80  


Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys 
                85                  90                  95      


Val Thr Leu Asn Cys Lys Asp Val Asn Ala Thr Asn Thr Thr Asn Asp 
            100                 105                 110         


Ser Glu Gly Thr Met Glu Arg Gly Glu Ile Lys Asn Cys Ser Phe Asn 
        115                 120                 125             


Ile Thr Thr Ser Ile Arg Asp Glu Val Gln Lys Glu Tyr Ala Leu Phe 
    130                 135                 140                 


Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asn Asn Thr Ser Tyr Arg 
145                 150                 155                 160 


Leu Ile Ser Cys Asp Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Ile 
                165                 170                 175     


Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala 
            180                 185                 190         


Ile Leu Lys Cys Asn Asp Lys Thr Phe Asn Gly Lys Gly Pro Cys Lys 
        195                 200                 205             


Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser 
    210                 215                 220                 


Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val Val Ile 
225                 230                 235                 240 


Arg Ser Asp Asn Phe Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu 
                245                 250                 255     


Lys Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 
            260                 265                 270         


Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly Glu 
        275                 280                 285             


Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Arg Ala Lys 
    290                 295                 300                 


Trp Asn Asp Thr Leu Lys Gln Ile Val Ile Lys Leu Arg Glu Gln Phe 
305                 310                 315                 320 


Glu Asn Lys Thr Ile Val Phe Asn His Ser Ser Gly Gly Asp Pro Glu 
                325                 330                 335     


Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn 
            340                 345                 350         


Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Asn Asn Thr Glu Gly Ser 
        355                 360                 365             


Asn Asn Thr Glu Gly Asn Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln 
    370                 375                 380                 


Ile Ile Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro 
385                 390                 395                 400 


Ile Arg Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu 
                405                 410                 415     


Thr Arg Asp Gly Gly Ile Asn Glu Asn Gly Thr Glu Ile Phe Arg Pro 
            420                 425                 430         


Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 
        435                 440                 445             


Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Cys Lys 
    450                 455                 460                 


Arg Arg Val Val Gln Arg Arg Arg Arg Arg Arg Ala Val Gly Ile Gly 
465                 470                 475                 480 


Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala 
                485                 490                 495     


Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile 
            500                 505                 510         


Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Pro Glu Ala Gln Gln Arg 
        515                 520                 525             


Met Leu Lys Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val 
    530                 535                 540                 


Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln Gln Leu Leu Gly Ile Trp 
545                 550                 555                 560 


Gly Cys Ser Gly Lys Leu Ile Cys Cys Thr Ala Val Pro Trp Asn Ala 
                565                 570                 575     


Ser Trp Ser Asn Lys Ser Leu Asp Arg Ile Trp Asn Asn Met Thr Trp 
            580                 585                 590         


Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr Ser Glu Ile Tyr Thr 
        595                 600                 605             


Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu 
    610                 615                 620                 


Leu Glu Leu Asp Ala Ala Ala Trp Ser His Pro Gln Phe Glu Lys Gly 
625                 630                 635                 640 


Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln 
                645                 650                 655     


Phe Glu Lys 
            


<210>  11
<211>  665
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial protein sequence of Strep-Tagged BG505 
       SOSIP(1-5).R6.664 gp140 comprising an engineered HIV-1 clade A 
       BG505 gp140, a peptide linker, and a Strep-Tag.

<400>  11

Ala Glu Asn Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 
1               5                   10                  15      


Asp Ala Glu Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu 
            20                  25                  30          


Thr Glu Lys His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp 
        35                  40                  45              


Pro Asn Pro Gln Glu Ile His Leu Glu Asn Val Thr Glu Glu Phe Asn 
    50                  55                  60                  


Met Trp Lys Asn Asn Met Val Glu Gln Met His Thr Asp Ile Ile Ser 
65                  70                  75                  80  


Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys 
                85                  90                  95      


Val Thr Leu Gln Cys Thr Asn Val Thr Asn Asn Ile Thr Asp Asp Met 
            100                 105                 110         


Arg Gly Glu Leu Lys Asn Cys Ser Phe Asn Met Thr Thr Glu Leu Arg 
        115                 120                 125             


Asp Lys Lys Gln Lys Val Tyr Ser Leu Phe Tyr Arg Leu Asp Val Val 
    130                 135                 140                 


Gln Ile Asn Glu Asn Gln Gly Asn Arg Ser Asn Asn Ser Asn Lys Glu 
145                 150                 155                 160 


Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile Thr Gln Ala Cys Pro 
                165                 170                 175     


Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly 
            180                 185                 190         


Phe Ala Ile Leu Lys Cys Lys Asp Lys Lys Phe Asn Gly Thr Gly Pro 
        195                 200                 205             


Cys Pro Ser Val Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro Val 
    210                 215                 220                 


Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Val 
225                 230                 235                 240 


Met Ile Arg Ser Glu Asn Ile Thr Asn Asn Ala Lys Asn Ile Leu Val 
                245                 250                 255     


Gln Phe Asn Thr Pro Val Gln Ile Asn Cys Thr Arg Pro Asn Asn Asn 
            260                 265                 270         


Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala Thr 
        275                 280                 285             


Gly Asp Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Val Ser Lys 
    290                 295                 300                 


Ala Thr Trp Asn Glu Thr Leu Gly Lys Val Val Lys Gln Leu Arg Lys 
305                 310                 315                 320 


His Phe Gly Asn Asn Thr Ile Ile Arg Phe Ala Asn Ser Ser Gly Gly 
                325                 330                 335     


Asp Leu Glu Val Thr Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe 
            340                 345                 350         


Tyr Cys Asn Thr Ser Gly Leu Phe Asn Ser Thr Trp Ile Ser Asn Thr 
        355                 360                 365             


Ser Val Gln Gly Ser Asn Ser Thr Gly Ser Asn Asp Ser Ile Thr Leu 
    370                 375                 380                 


Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Arg Ile Gly Gln 
385                 390                 395                 400 


Ala Met Tyr Ala Pro Pro Ile Gln Gly Val Ile Arg Cys Val Ser Asn 
                405                 410                 415     


Ile Thr Gly Leu Ile Leu Thr Arg Asp Gly Gly Ser Thr Asn Ser Thr 
            420                 425                 430         


Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg 
        435                 440                 445             


Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val 
    450                 455                 460                 


Ala Pro Thr Arg Cys Lys Arg Arg Val Val Gly Arg Arg Arg Arg Arg 
465                 470                 475                 480 


Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala 
                485                 490                 495     


Gly Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg 
            500                 505                 510         


Asn Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala 
        515                 520                 525             


Pro Glu Ala Gln Gln His Leu Leu Lys Leu Thr Val Trp Gly Ile Lys 
    530                 535                 540                 


Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Arg Asp Gln 
545                 550                 555                 560 


Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Cys Thr 
                565                 570                 575     


Asn Val Pro Trp Asn Ser Ser Trp Ser Asn Arg Asn Leu Ser Glu Ile 
            580                 585                 590         


Trp Asp Asn Met Thr Trp Leu Gln Trp Asp Lys Glu Ile Ser Asn Tyr 
        595                 600                 605             


Thr Gln Ile Ile Tyr Gly Leu Leu Glu Glu Ser Gln Asn Gln Gln Glu 
    610                 615                 620                 


Lys Asn Glu Gln Asp Leu Leu Ala Leu Asp Ala Ala Ala Trp Ser His 
625                 630                 635                 640 


Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser 
                645                 650                 655     


Ala Trp Ser His Pro Gln Phe Glu Lys 
            660                 665 


<210>  12
<211>  659
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial protein sequence of Strep-Tagged SF162 SOSIP.R6.664 
       gp140 comprising an engineered HIV-1 clade B SF162 gp140, a 
       peptide linker, and a Strep-Tag.

<400>  12

Val Glu Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys 
1               5                   10                  15      


Glu Ala Thr Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp 
            20                  25                  30          


Thr Glu Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp 
        35                  40                  45              


Pro Asn Pro Gln Glu Ile Val Leu Glu Asn Val Thr Glu Asn Phe Asn 
    50                  55                  60                  


Met Trp Lys Asn Asn Met Val Glu Gln Met His Glu Asp Ile Ile Ser 
65                  70                  75                  80  


Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys 
                85                  90                  95      


Val Thr Leu His Cys Thr Asn Leu Lys Asn Ala Thr Asn Thr Lys Ser 
            100                 105                 110         


Ser Asn Trp Lys Glu Met Asp Arg Gly Glu Ile Lys Asn Cys Ser Phe 
        115                 120                 125             


Lys Val Thr Thr Ser Ile Arg Asn Lys Met Gln Lys Glu Tyr Ala Leu 
    130                 135                 140                 


Phe Tyr Lys Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr Ser Tyr 
145                 150                 155                 160 


Lys Leu Ile Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys 
                165                 170                 175     


Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe 
            180                 185                 190         


Ala Ile Leu Lys Cys Asn Asp Lys Lys Phe Asn Gly Ser Gly Pro Cys 
        195                 200                 205             


Thr Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val 
    210                 215                 220                 


Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Gly Val Val 
225                 230                 235                 240 


Ile Arg Ser Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln 
                245                 250                 255     


Leu Lys Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr 
            260                 265                 270         


Arg Lys Ser Ile Thr Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly 
        275                 280                 285             


Asp Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Ile Ser Gly Glu 
    290                 295                 300                 


Lys Trp Asn Asn Thr Leu Lys Gln Ile Val Thr Lys Leu Gln Ala Gln 
305                 310                 315                 320 


Phe Gly Asn Lys Thr Ile Val Phe Lys Gln Ser Ser Gly Gly Asp Pro 
                325                 330                 335     


Glu Ile Val Met His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys 
            340                 345                 350         


Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Asn Asn Thr Ile Gly Pro 
        355                 360                 365             


Asn Asn Thr Asn Gly Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile 
    370                 375                 380                 


Ile Asn Arg Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile 
385                 390                 395                 400 


Arg Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu Leu Thr 
                405                 410                 415     


Arg Asp Gly Gly Lys Glu Ile Ser Asn Thr Thr Glu Ile Phe Arg Pro 
            420                 425                 430         


Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr 
        435                 440                 445             


Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro Thr Lys Cys Lys 
    450                 455                 460                 


Arg Arg Val Val Gln Arg Arg Arg Arg Arg Arg Ala Val Thr Leu Gly 
465                 470                 475                 480 


Ala Met Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala 
                485                 490                 495     


Arg Ser Leu Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile 
            500                 505                 510         


Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Pro Glu Ala Gln Gln His 
        515                 520                 525             


Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val 
    530                 535                 540                 


Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly Ile Trp 
545                 550                 555                 560 


Gly Cys Ser Gly Lys Leu Ile Cys Cys Thr Ala Val Pro Trp Asn Ala 
                565                 570                 575     


Ser Trp Ser Asn Lys Ser Leu Asp Gln Ile Trp Asn Asn Met Thr Trp 
            580                 585                 590         


Met Glu Trp Glu Arg Glu Ile Asp Asn Tyr Thr Asn Leu Ile Tyr Thr 
        595                 600                 605             


Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu 
    610                 615                 620                 


Leu Glu Leu Asp Ala Ala Ala Trp Ser His Pro Gln Phe Glu Lys Gly 
625                 630                 635                 640 


Gly Gly Ser Gly Gly Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln 
                645                 650                 655     


Phe Glu Lys 
            


<210>  13
<211>  6
<212>  PRT
<213>  Artificial Sequence

<220>
<223>  Artificial enhanced furin cleavage site protein sequence

<400>  13

Arg Arg Arg Arg Arg Arg 
1               5       


