Expression of CRLAAO in E.coli

Attempts were made to clone and express CRLAAO inorder to obtain recombinant unglycosylated CRLAAO. Unless mentioned, CRLAAOS^- region was PCR amplified from pTOPOCRLAAO^® and sub-cloned into various expression vectors as mentioned below.

2.4.3.1 Construction of pET16bCRLAAO plasmid.

The expression vector pET16b was digested with NdeI and BamHI endonucleases. For the insert preparation, CRLAAOS^- cDNA coding region was released with corresponding restriction enzymes from the intermediate TOPO^® plasmid. After the ligation, E.coli transformation the plasmid was sequenced and the integration of CRLAAO cDNA fragment was verified by alignment as described in the Methods section. The sub-cloning of CRLAAO cDNA fragment from TOPO vector into the pET16b expression vector is represented in Fig. 7.

Fig. 7. Restriction analysis of the pET16bLAAOS^- construction. Lanes 1 and 11: λ HinDIII/

BamHI DNA marker (NEB). Lanes 2, 3, 4 and 5: pET16b undigested, NdeI digest, BamHI digest and double digests respectively. Lane 6 and 16: 2 µL each of 100 bp ladder (NEB).

Lanes 7, 8, 9 and 10: pTOPO^®LAAOS^- undigested, NdeI digest, BamHI digest and double digests, respectively. First and second arrows represent the fragments of vector and insert respectively. Lanes 12, 13, 14 and 15: pET16bLAAOS^- undigested, NdeI digest, BamHI digest and double digests respectively

2.4.3.2 Expression of His10CRLAAO in BL21DE3 E.coli

For expression of unglycosylated CRLAAO with N-terminal His10-tag, pET16bCRLAAOS^- construct was transformed into BL21DE3 competent cells as described in the Methods section. Expression of CRLAAO is under the control of the T7 promoter. T7 RNA polymerase gene is under the lacUV5 promoter, which is inducible by IPTG. Addition of IPTG to a growing culture of the lysogen induces T7 RNA polymerase production, which in turn transcribes the target DNA in the plasmid. An outline of pET expression system is shown in Fig. 8. Inorder to estimate an optimum IPTG concentration for CRLAAO expression, induction of protein expression was performed at various IPTG concentrations, E.coli cultures were grown to an OD600 = 1. Then they were induced with different concentrations of IPTG, grown for 4 more hours and then harvested. Cell lysates were analysed on 12% SDS PAGE (Fig. 9). It should be stated that the induction of expression was observed even without IPTG concentration. This could be attributed to the fact that the genes cloned under lac promoter show leaky expression.

Fig. 8. Expression in BL21DE3 E.coli cells. T7 RNA polymerase is a gene product of the T7gene I encoded by chromosomal λ-lysogen (DE3). The T7 geneI is under control of the lac promoter. The Lac-Operon is regulated by the Lac-promoter and hence the overexpression of CRLAAO can be provoked in the presence of IPTG as an inducer. (Source: pET system manual, Novagen)

Fig. 9. Optimization of IPTG induction. E.coli cultures induced with the indicated concentrations of IPTG after OD600 reached 1. Total cell lysates (200 µL) from 1 mL cultures were prepared by sonication and 10 µL of each sample was analyzed on 12% SDS PAGE.

(L): PNGase-F treated snake venom CRLAAO was used as positive control. (M): Protein markers (NEB broad range, P7708L).

2.4.3.3 Co-overexpression of CRLAAOS^- and chaperonins

Competent cells of E.coli harbouring pET16b-LAAOS^- were transformed with pGroESL plasmid DNA and the transformants were selected and maintained in medium containing ampicillin and chloramphenicol. Arabinose (0.02%) was used for the induction of expression of chaperonins EL & ES. Expression of CRLAAOS^- was enhanced in the presence of chaperonins, which was quite evident from the fact that CRLAAOS^- expression levels in the absence of chaperonins at 5 mM IPTG (Fig. 9) were comparable with that of 0.1mM IPTG in the presence of chaperonins GroES and GroEL (Fig. 10 A). From the SDS PAGE (Fig. 10 B) and Western blot (Fig. 10 C) using αHis10 antibodies it was demonstrated that the solubility could be improved when the cultures were grown at low temperatures. Here the protein concentrations were normalized using standard Bradford assay. Thus for expressing CRLAAO in the soluble form, E.coli cultures were grown at 25 °C with 0.02% arabinose and 1 mM IPTG was included during the induction of protein expression. Induction was generally maintained until the cell growth reached saturation (OD600= 6-8 or over night induction). Cross reactivity of CRLAAO antibodies developed against the native snake venom further confirmed that the recombinant CRLAAO is correctly expressed. Additional 22 amino acids were added to the N-terminal end of the mature CRLAAO sequence. A stretch of amino acids including a sequence of 10 consecutive histidines (His10-tag) and a spacer connecting this His-tag to the CRLAAO sequence added 6.7 kDa to the nascent CRLAAO (55 kDa). Thus the molecular size of the expressed recombinant unglycosylated CRLAAO is nearly 62 kDa. This was also demonstrated on SDS PAGE as the glycosylated CRLAAO (control) has the same electrophoretic mobility as the expressed CRLAAO. Since the cultures were induced with 0.02% arabinose before IPTG induction, chaperonin GroEL induction was maintained all the time. The chaperonin GroEL has a slightly lower mobility than the expressed CRLAAO. As a negative control the cultures of E.coli transformed with vector alone and without chaperonin expressing plasmids were used. No induction of either of the proteins could be detected on SDS PAGE.

Native CRLAAO treated with PNGase-F was used as control for unglycosylated native CRLAAO (55 kDa).

Fig. 10. Optimization of CRLAAOS^- expression. A Total cell lysates of BL21DE3 E.coli containing pGroESL and pET16bLAAOS^- plasmids were induced with Arabinose and IPTG.

Cell lysates were analyzed on 12% SDS PAGE. B Cultures were induced with 5mM IPTG and grown at indicated temperatures. Soluble and insoluble fractions were separated by sonication and analyzed on 12% SDS PAGE. C. CRLAAO expression was detected by αHis antibodies on Western blot. (U): uninduced; (I): induced; (S): soluble; (P): Pellet, insoluble;

(L) unglycosylated CRLAAO control. (V): BL21DE3 transformed with pET16b vector as negative control. (M): Protein marker, NEB low range.

2.4.3.4 Construction of pBADCRLAAO-Trx plasmid (N-terminal thioredoxin fusion)

To express CRLAAO with an N-terminal thioredoxin fusion, the cDNA of CRLAAO was cloned into the pBAD TOPO expression vector. The TOPO cloning method is schematically shown in Fig. 11A. The ligation mixture was transformed into Top 10 chemically competent E.coli cells and the transformants were selected on kanamycin containing medium. Several clones positive for the presence of CRLAAO were selected by colony PCR. Fig. 11 B shows the plasmid map of the resultant clone. The correct orientation of the PCR insert was verified by restriction analysis (Fig. 11) and sequencing.

Fig. 11. Construction of pBADCRLAAO plasmid clone. A. Directional cloning of the PCR product. The overhang in the cloning vector (GTGG) invades the 5´ end of the PCR product, anneals to the added bases, and stabilizes the PCR product in the correct orientation.(Source:

pBAD instruction manual; cat. 062602, Invitrogen) B. Plasmid map showing the N-terminal thioredoxin in translational fusion with CRLAAO. C. Restriction analysis. Lanes 1, 2 and 3 are pBADCRLAAO undigested, NdeI and ApaI digested respectively. Lane 4 shows the fragment (1.4 kbp) released upon PstI digestion. Second PstI site (2176) in the clone confirms the presence of PCR insert. Lane 5, λ DNA Marker (HindIII/EcoRI).

2.4.3.5 Optimization of CRLAAO-Trx expression

LMG194 strain of E.coli was used for optimisation of CRLAAO expression.

Cell lysates from the uninduced and induced cultures were analyzed by Western blotting. 0.02% arabinose concentration was optimal for obtaining maximum expression (Fig. 12 A). The optimum temperature for fermentation was determined by shaker cultures. After sub-culturing overnight grown E.coli LMG194-pBadLAAO to A600nm 0.1and grown at various temperatures. Cultures were induced with 0.02%

arabinose after the cell densities reached an OD600nm of 0.8. After saturation, the cell lysates were analyzed on 12% SDS PAGE and Western blotting. As shown in Fig. 12 B, nearly 5% of total expressed CRLAAO-Trx was found to be soluble when grown at 25 °C. Therefore further experiments were always carried out at 25ºC.

Fig. 12. Optimization of CRLAAOTrx expression. Western blots were performed using Rabbit αLAAO antibodies. A. Effect of arabinose concentration on CRLAAOTrx expression.

B. Effect of temperature on solubility of expressed CRLAAOTrx. (+): Positive control, native CRLAAO; (W): Whole cell lysate; (S): Soluble fraction; (P): Pellet, insoluble fraction.

2.4.3.6 Expressed CRLAAOTrx is insoluble.

For separating soluble and insoluble fractions, the recombinant LMG Ecoli were grown at 25 °C to log phase and the protein expression was induced with 0.02%

arabinose. After 4 h of induction period, cells were harvested and the soluble and insoluble fractions of the cell lysate were prepared as mentioned in the Methods section. Samples were analyzed on 12% SDS PAGE. Most of the expressed protein was found in the inclusion bodies (Fig. 13). Unglycosylatd CRLAAO was used as positive control.

Fig. 13. Expressed CRLAAOTrx is highly insoluble. After induction, soluble and insoluble fractions were separated and analyzed on 12% SDS PAGE. Almost all the expressed protein is in found in insoluble fraction of the cell lysate. (M) Protein standards, NEB broad range;

(U): Un induced; (L): Native CRLAAO; (W): Whole cell lysate; (S): Soluble fraction; (P):

Pellet, insoluble fraction

2.4.3.7 Co-expression of CRLAAOTrx and chaperonins

Inorder to enhance the solubility of expressed CRLAAOTrx, co-overexpression of CRLAAOTrx and chaperonins was attempted. Competent cells of recombinant LMGLAAOTrx E.coli were transformed with pGroESL and the transformants were maintained on kanamycin containing medium. 0.02% arabinose was used to induce CRLAAOTrx expression since the chaperonins EL and ES are under arabinose induction. After induction, the soluble and insoluble fractions of the cell lysate were analyzed on 12% SDS PAGE (Fig. 14). It was evident that the solubility could not be improved to such levels that could be detected on SDS PAGE.

Thus either addition of N-terminal thioredoxin or co-over expression with the chaperonins did not help improving the solubility of CRLAAO.

Fig. 14. Co-over expression of CRLAAOTrx and chaperonins. After induction, soluble and insoluble fractions were separated and analyzed on 12% SDS PAGE.

Almost all the expressed protein was found in the insoluble fraction of the cell lysate. (W): Whole cell lysate; (S):

Soluble fraction; (P): Pellet, insoluble fraction.

2.4.3.8 Purification of His10CRLAAO from E.coli

10 g E.coli wet paste was used for the purification of soluble His tagged CRLAAO. After applying the soluble fraction of the cell lysate on a 1 mL Ni-NTA column, an imidazol gradient was applied as shown in the Fig. 15. The purified fraction (F53) was analyzed on 12% SDS PAGE (Fig. 15 inset). Yields of soluble recombinant CRLAAO were typically between 0.15 - 0.2 mg/L of E.coli culture. The soluble His10CRLAAO did not show activity even after the attempts of reconstitution with FAD (see Methods section). Due to the insolubility problems, eukaryotic expression systems were tried inorder to obtain the active soluble CRLAAO.

Fig. 15. Purification of soluble His10LAAO from E.coli. Elution profile of purification of expressed CRLAAO from E.coli from the soluble fraction of the cell lysate. Inset shows the purified protein eluted (F53): Fraction no. 53; (M): Protein standards; (AU): Absorbance.