Nuevas competencias para el profesor y el estudiante universitario
1. La universidad de Antioquia requiere nuevas competencias
4.1 ZZ-PhaC to ZZ-Linker-ZZ-PhaC
The ZZ domain of protein A is a well-established example of a successful and functional covalent attachment to the PHA synthase and thus the PHA granule surface. At the onset of this study, we proposed that the ideal target for attachment and subsequent display of bacterial immunoglobulin binding proteins such as protein G and L would be to the C- terminus of PhaC, thus creating a fusion protein displaying dual functionality on a single platform without any additional cost. A functional construct from a previous study, pET-14b- ZZ-phaC-GFP, was an ideal starting point. The GFP fragment could be excised via restriction endonuclease digest and the respective gene encoding for a desired protein designed with identical restriction endonuclease cut-sites to the excised fragment could be digested and subsequently ligated with the construct, thus replacing GFP with the gene of interest.
However, immunoglobulin binding experiments with the resulting pET-14b-ZZ-phaC-GB13 indicated poor binding efficiency. Most of the IgG was found in the unbound fraction, with less than 50% of added IgG obtained in the elution fraction, indicating poor binding both with IgG from human serum and IgG from goat serum. This changed in 2011 when a new construct was generated, linking 2 separate ZZ domains with a linker region to produce ZZ- Linker-ZZ-PhaC. The importance of this linker region was described in a previous study (Jahns & Rehm, 2009). The effect of the described linker on the AG-bead construct was then tested. A human IgG binding assay with 3mg IgG from human serum was added to 50mg of each type of bead. The protein concentration of the elution fractions obtained is indicated below.
Figure 15: Protein concentration of elution fractions obtained from binding experiment of 50mg PHA beads with 3mg IgG from human serum.
The increase in binding efficiency (Figure 15) seen in the new ZZ-linker-ZZ-PhaC-GB13 construct (49.7% increase in eluted protein concentration) further confirms that the addition of the linker region facilitates the correct display of proteins on the surface of the PHA
granule while maintaining the hydrophobic environment close to the synthase (Jahns &Rehm, 2009). The linker is not necessary for the C terminal fusion of GB13 to PhaC. As in the case of GFP, the length of the fusion protein and the confirmed functionality of the GB13 domain suggest that it inherently maintains the hydrophobic region required for the synthase to remain active. The ZZ-Linker-ZZ-PhaC fragment was therefore used for the generation of AG- and AL-fusion beads.
0.000 0.200 0.400 0.600 0.800 1.000 1.200 ZZ-phaC-GB13 ZZ-L-ZZ-phaC-GB13 Pr o te in C o n ce n tr ation (m g/ m l) Bead type
4.2 ZZ-Linker-ZZ-PhaC-L
As mentioned in Chapter 3 (3.2), the analysis of the AL-fusion bead by SDS-PAGE yielded unexpected results. The fusion protein obtained was not of the expected size.
Figure 16: SDS-PAGE analysis of isolated and purified AL-beads. The expected fusion protein size of ZZ-Linker-ZZ-PhaC-L is 142kDa. Wild-type PhaC expected size is 64KdA.
As seen in the 8% SDS-PAGE above, there is no intense band signifying overexpression at 142kDa. Instead, a band suggesting overexpression is seen at 97kDa. The beads were subsequently harvested and purified once more; however, the same result was obtained.
The DNA sequence of the construct used to transform the production strain (pET14b-ZZ- linker-ZZ-phaC-L) was re-sequenced and checked for possible errors such as previously unnoticed stop codons. No such errors were found. Protease inhibitor tablets were then used during downstream processing to rule out the possibility that the fusion protein was being cleaved during the purification process.
Figure 17: SDS-PAGE of isolated and purified AL-beads with and without the addition or protease inhibitor. Expected molecular weights are indicated. PHA beads run in lane 3 were processed in 50 mM potassium phosphate buffer (pH 7.5) and 1 tablet of Complete Mini EDTA-free protease inhibitor cocktail (Roche, Switzerland). PHA beads run in lane 4 were processed without the protease inhibitor being added. Arrows indicate fragments resulting from the cleavage of the fusion protein.
The addition of protease inhibitor did not alter the unexpected result, and the fusion protein of the expected size was still not expressed. The cleavage of the 142 kDA fusion protein is clearly seen in the SDS-PAGE above. We hypothesize that the 142 kDa fusion protein was cleaved into 2 separate fragments of 97 kDa and 45 kDa as indicated by the overexpressed band at those molecular weight marks. This initially seemed to correspond with 2 separate parts of the fusion protein, the 97 kDa fragment matching the estimated molecular weight of ZZ-linker-ZZ-PhaC and the 45 kDa fragment matching the L-domain protein. To assess if
either of the proteins were still functional, we performed an IgG binding assay using the purified ZZ-linker-ZZ-PhaC-L beads.
Results from immunoglobulin binding experiments with both IgG from rat serum and human serum suggested that neither the L domain nor the ZZ domains of the fusion protein were functional, suggesting that the proteins were inactive and most likely misfolded.
In order to display both domains on a single construct, we then proposed and partially executed a method involving the use of Polymerase Chain Reaction (PCR) to produce a fusion protein with the L-domain displayed on the N-terminus of PhaC instead of the C- terminus. This is achieved by introducing new restriction endonuclease cutsites (NdeI) flanking the L-domain synthetic gene thus allowing for subsequent ligation to the 5’ end of the phaC gene. Similarly, protein A’s ZZ domain would be displayed on the C-terminus of PhaC connected to the synthase by a linker region (Jahns & Rehm, 2009). However, due to time constraints, this experiment was not included in this particular study and will be pursued as future work on the topic.
4.3 Summary
The performance of the novel, engineered protein G, protein L and protein AG-fusion PHA bead met the expectations we set in the hypothesis of this study. Through the methodology above we have demonstrated the ability to display these proteins on the surface of a bio polyester bead produced in recombinant E.coli using PHA synthase immobilization technology. The ability to display 2 different immunoglobulin binding proteins derived from different bacteria on a single platform greatly increases the value of the novel PHA bead by adding functionality and binding efficiency without increasing the cost it would require to produce a single-function bead. The tests of functionality conducted on the novel protein L
PHA beads also suggests that these beads could serve as an critical tool for purification of immunoglobulin from serums and hybridoma supernatants.
In this study, we have confirmed that it is possible to express multiple binding domains on the surface of the PHA granule while maintaining functionality (and even improving on binding performance set by commercial standards). This in turn opens up many other possibilities, such as the creation of beads displaying combinations of other binding domains. The convenient, one-step cultivation and subsequent purification of PHA granules would mean that these highly-specific affinity purification tools could be produced on-demand on a large scale. The process of eluting the bound immunoglobulin from the beads does not have an adverse effect on the epitopes of the immunoglobulin molecule as demonstrated in the results of the lateral flow assay. There also exists the possibility of adding more functionality to the beads by expressing more domains via PHA synthase attachment and utilizing the linker sequence to maintain correct orientation and display of the binding domains. A “super- bead” with the binding domains of protein A, G, L and mannan-binding protein (MBP)
APPENDIX
GC/MS results (analysis performed by Plant & Food Research, NZ)
Data File Name PHB Std - 2mg
Peak# Ret.Time Area Name
1 6.297 721002 Cyclotrisiloxane, hexamethyl-
2 7.513 66429775 methyl 3-hydroxybutanoate
3 7.706 2699686 Sulfuric acid, dimethyl ester
4 8.728 4261062 2-Propanol, 1- 1-methyl-2-(2-propenyloxy)ethoxy -
5 31.334 1096806
6 34.778 559909
7 36.125 2249921 Hexadecanoic acid, methyl ester
8 38.14 826300
9 39.957 525580
10 40.443 962605
Data File Name PHB Std - 4mg
Peak# Ret.Time Area Name
1 7.513 141794326 methyl 3-hydroxybutanoate
2 7.667 2877365 Sulfuric acid, dimethyl ester
3 36.142 2391490 methyl hexadecanoate, C16:0 Me ester
Data File Name PHB Std - 10mg
Peak# Ret.Time Area Name
1 7.623 378137860 methyl 3-hydroxybutanoate
2 36.112 7626342 Hexadecanoic acid, methyl ester
3 38.095 696828 Octadecanoic acid, methyl ester
Data File Name PHB Std - 15mg
Peak# Ret.Time Area Name
1 7.675 504407324 methyl 3-hydroxybutanoate
2 36.103 11316068 methyl hexadecanoate, C16:0 Me ester
3 37.883 621469
4 38.087 1073081 methyl octadecanoate, C18:0 Me ester
Data File Name ZZ-Linker-ZZ-PhaC-GB13
Peak# Ret.Time Area Name
1 7.57 202016426 methyl 3-hydroxybutanoate
2 7.694 1750301 Sulfuric acid, dimethyl ester
3 8.688 9213489 2-Propanol, 1- 1-methyl-2-(2-propenyloxy)ethoxy -
4 32.648 4142081 Tetradecanoic acid, methyl ester
5 35.833 9968489 9-Hexadecenoic acid, methyl ester, (Z)-
6 35.958 544148
7 36.099 32147925 methyl hexadecanoate, C16:0 Me ester
8 37.544 508030
9 37.907 18815567 C18:1n9c FAME - oleic
10 38.079 1012360 methyl octadecanoate, C18:0 Me ester
Data File Name ZZ-Linker-ZZ-PhaC-L
Peak# Ret.Time Area Name
1 7.582 255823905 methyl 3-hydroxybutanoate
2 7.687 1705396 Sulfuric acid, dimethyl ester
3 32.649 3669269 C14:0 Me ester
4 35.829 10409365 9-Hexadecenoic acid, methyl ester, (Z)-
5 35.95 896471
6 36.096 36265493 methyl hexadecanoate, C16:0 Me ester
7 37.541 597728
8 37.906 10287996 C18:1n9t FAME - elaidic
9 38.078 1228365 Octadecanoic acid, methyl ester
10 39.119 563140
11 39.376 559563
Data File Name PHB Std - 2mg
Peak# Ret.Time Area Name
1 7.468 47903345 methyl 3-hydroxybutanoate
2 7.673 1761061 Sulfuric acid, dimethyl ester
3 8.702 2793341 Propanoic acid, 2-hydroxy-2-methyl-, ethyl ester
4 36.159 1569662 Hexadecanoic acid, methyl ester
Data File Name PHB Std - 4mg
Peak# Ret.Time Area Name
1 7.535 112084628 methyl 3-hydroxybutanoate
2 7.694 2409628 Sulfuric acid, dimethyl ester
3 8.705 6199633 2-Propanol, 1- 1-methyl-2-(2-propenyloxy)ethoxy -
4 36.16 2028951 methyl hexadecanoate, C16:0 Me ester
Data File Name PHB Std - 10mg
Peak# Ret.Time Area Name
1 7.632 305942071 methyl 3-hydroxybutanoate
2 7.717 736938 Sulfuric acid, dimethyl ester
3 8.695 16234249 2-Propanol, 1- 1-methyl-2-(2-propenyloxy)ethoxy -
4 36.136 5131962 methyl hexadecanoate, C16:0 Me ester
5 38.107 525558 Octadecanoic acid, methyl ester
Data File Name PHB Std - 15mg
Peak# Ret.Time Area Name
1 7.693 430232870 methyl 3-hydroxybutanoate
2 8.702 22375108 2-Propanol, 1- 1-methyl-2-(2-propenyloxy)ethoxy -
3 36.127 8077232 Hexadecanoic acid, methyl ester
4 38.108 762105 Octadecanoic acid, methyl ester
Sample Area Weight (mg) PHB %PHB/mg
ZZ-Linker-ZZ-PhaC-GB13 202016426 15 6.72 44.82
MALDI-TOF (analysis performed by The Centre for Protein Research, Otago University)
ZZ-linker-ZZ-PhaC-GB13
Protein View
Match to: Seq2_10081 Score: 2692
(10081_Seq2) K2
Found in search of 10081_K2_combined.txt
Nominal mass (Mr): 103690; Calculated pI value: 5.29
NCBI BLAST search of Seq2_10081 against nr
Unformatted sequence string for pasting into other applications
Variable modifications: Carbamidomethyl (C),Deamidation (NQ),Oxidation (M),Pyro-glu (N-term E),Pyro-glu (N-term Q)
Cleavage by Trypsin: cuts C-term side of KR unless next residue is P
Sequence Coverage: 53%
Matched peptides shown in Bold Red
1 MAQHDEAVDN KFNKEQQNAF YEILHLPNLN EEQRNAFIQS LKDDPSQSAN
51 LLAEAKKLND AQAPKVDNKF NKEQQNAFYE ILHLPNLNEE QRNAFIQSLK
101 DDPSQSANLL AEAKKLNDAQ APKVDANSSS VPHMATGKGA AASTQEGKSQ
151 PFKVTPGPFD PATWLEWSRQ WQGTEGNGHA AASGIPGLDA LAGVKIAPAQ
201 LGDIQQRYMK DFSALWQAMA EGKAEATGPL HDRRFAGDAW RTNLPYRFAA
251 AFYLLNARAL TELADAVEAD AKTRQRIRFA ISQWVDAMSP ANFLATNPEA
301 QRLLIESGGE SLRAGVRNMM EDLTRGKISQ TDESAFEVGR NVAVTEGAVV
351 FENEYFQLLQ YKPLTDKVHA RPLLMVPPCI NKYYILDLQP ESSLVRHVVE
401 QGHTVFLVSW RNPDASMAGS TWDDYIEHAA IRAIEVARDI SGQDKINVLG
451 FCVGGTIVST ALAVLAARGE HPAASVTLLT TLLDFADTGI LDVFVDEGHV
501 QLREATLGGG AGAPCALLRG LELANTFSFL RPNDLVWNYV VDNYLKGNTP
551 VPFDLLFWNG DATNLPGPWY CWYLRHTYLQ NELKVPGKLT VCGVPVDLAS
601 IDVPTYIYGS REDHIVPWTA AYASTALLAN KLRFVLGASG HIAGVINPPA
651 KNKRSHWTND ALPESPQQWL AGAIEHHGSW WPDWTAWLAG QAGAKRAAPA
701 NYGNARYRAI EPAPGRYVKA KALEVLAVID KRGGGGGSGG GSGGGSGGGG
751 SKTDTYKLIL NGKTLKGETT TEAVDAATAE KVFKQYANDN GVDGEWTYDD
801 ATKTFTVTEK PESGGGSGGG SGGGGSKTDT YKLILNGKTL KGETTTEAVD
851 AATAEKVFKQ YANDNGVDGE WTYDDATKTF TVTEKPESGG GSGGGSGGGG
901 SKTDTYKLIL NGKTLKGETT TEAVDAATAE KVFKQYANDN GVDGEWTYDD
951 ATKTFTVTEK PE
Sort Peptides By
Residue Number Increasing Mass Decreasing Mass
Start - End Observed Mr(expt) Mr(calc) Delta Miss Sequence
15 - 34 2485.2549 2484.2476 2484.2033 0.0443 0 K.EQQNAFYEILHLPNLNEEQR.N (Ions score 143)
35 - 56 2360.2366 2359.2293 2359.2019 0.0274 1 R.NAFIQSLKDDPSQSANLLAEAK.K (Ions score 84)
154 - 169 1858.9371 1857.9298 1857.9050 0.0249 0 K.VTPGPFDPATWLEWSR.Q (Ions score 116)
196 - 207 1309.7419 1308.7346 1308.7150 0.0197 0 K.IAPAQLGDIQQR.Y (Ions score 95)
211 - 223 1453.6975 1452.6902 1452.6707 0.0195 0 K.DFSALWQAMAEGK.A (Ions score 74)
211 - 223 1469.6899 1468.6826 1468.6656 0.0170 0 K.DFSALWQAMAEGK.A Oxidation (M) (Ions score 24)
224 - 233 1066.5544 1065.5471 1065.5203 0.0268 0 K.AEATGPLHDR.R (Ions score 50)
248 - 258 1256.6993 1255.6920 1255.6713 0.0207 0 R.FAAAFYLLNAR.A (Ions score 84)
259 - 272 1416.7524 1415.7451 1415.7143 0.0308 0 R.ALTELADAVEADAK.T (Ions score 60)
279 - 302 2664.3423 2663.3350 2663.2802 0.0549 0 R.FAISQWVDAMSPANFLATNPEAQR.L (Ions score 168)
279 - 302 2680.3162 2679.3089 2679.2751 0.0339 0 R.FAISQWVDAMSPANFLATNPEAQR.L Oxidation (M) (Ions score 71)
303 - 313 1173.6736 1172.6663 1172.6400 0.0263 0 R.LLIESGGESLR.A (Ions score 84)
328 - 340 1438.7090 1437.7017 1437.6735 0.0282 0 K.ISQTDESAFEVGR.N (Ions score 107)
341 - 367 3115.6604 3114.6531 3114.5912 0.0619 0 R.NVAVTEGAVVFENEYFQLLQYKPLTDK.V (Ions score 100)
383 - 396 1695.9159 1694.9086 1694.8879 0.0207 0 K.YYILDLQPESSLVR.H (Ions score 102)
397 - 411 1793.9689 1792.9616 1792.9372 0.0244 0 R.HVVEQGHTVFLVSWR.N (Ions score 129)
412 - 432 2320.0588 2319.0515 2319.0226 0.0290 0 R.NPDASMAGSTWDDYIEHAAIR.A (Ions score 89)
412 - 432 2336.0583 2335.0510 2335.0175 0.0336 0 R.NPDASMAGSTWDDYIEHAAIR.A Oxidation (M) (Ions score 74)
439 - 468 3045.6870 3044.6797 3044.6327 0.0470 1
R.DISGQDKINVLGFCVGGTIVSTALAVLAAR.G Carbamidomethyl (C) (Ions score 112)
469 - 503 3750.0137 3749.0064 3748.9311 0.0753 0 R.GEHPAASVTLLTTLLDFADTGILDVFVDEGHVQLR.E (Ions score 131)
504 - 519 1513.8076 1512.8003 1512.7718 0.0285 0 R.EATLGGGAGAPCALLR.G Carbamidomethyl (C) (Ions score 128)
520 - 546 3200.7183 3199.7110 3199.6341 0.0769 0 R.GLELANTFSFLRPNDLVWNYVVDNYLK.G (Ions score 47)
547 - 575 3469.7136 3468.7063 3468.6389 0.0674 0 K.GNTPVPFDLLFWNGDATNLPGPWYCWYLR.H Carbamidomethyl (C) (Ions score 79)
576 - 584 1145.6191 1144.6118 1144.5876 0.0242 0 R.HTYLQNELK.V (Ions score 52)
576 - 584 1145.6198 1144.6125 1144.5876 0.0249 0 R.HTYLQNELK.V (Ions score 53)
612 - 631 2171.1375 2170.1302 2170.1058 0.0244 0 R.EDHIVPWTAAYASTALLANK.L (Ions score 108)
634 - 651 1748.0022 1746.9949 1746.9780 0.0169 0 R.FVLGASGHIAGVINPPAK.N (Ions score 103)
697 - 706 1004.5153 1003.5080 1003.4835 0.0245 0 R.AAPANYGNAR.Y (Ions score 53)
722 - 732 1226.7783 1225.7710 1225.7394 0.0317 1 K.ALEVLAVIDKR.G (Ions score 62)
767 - 781 1493.6984 1492.6911 1492.6893 0.0019 0 K.GETTTEAVDAATAEK.V (Ions score 43)
785 - 803 2144.9084 2143.9011 2143.8606 0.0405 0 K.QYANDNGVDGEWTYDDATK.T Pyro-glu (N-term Q) (Ions score 58)
785 - 803 2161.8940 2160.8867 2160.8872 -0.0005 0 K.QYANDNGVDGEWTYDDATK.T (Ions score 123)
804 - 827 2097.9668 2096.9595 2096.9609 -0.0014 0 K.TFTVTEKPESGGGSGGGSGGGGSK.T (Ions score 146)
954 - 962 1051.5243 1050.5170 1050.5233 -0.0063 0 K.TFTVTEKPE.- (Ions score 35)
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