대장균에서 재조합 단백질의 세포외 분비생산

Theories and Applications of Chem. Eng., 2002, Vol. 8, No. 1

정기준, 이상엽

한국과학기술원 생명화학공학과

Excretory production of recombinant proteins into culture medium by recombinant Escherichia coli

Ki Jun Jeong, Sang Yup Lee

화학공학의 이론과 응용 제8권 제1호 2002년

Dept. Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and Technology

서론

In Escherichia coli, the excretory production of recombinant proteins into the culture medium has several advantages: simple purification, improved protein folding, the least level of proteolysis and N-terminal authenticity, which are similar to those achievable by the secretion into the periplasm. However, excretion of recombinant proteins has other significant advantages compared with the secretion into the periplasm. There is no need to disrupt the outer cell membrane to recover the target proteins. It has been well known that osmotic shock method and other traditional methods to purify periplasmic proteins are difficult to scale-up. Furthermore, excretory production allows continuous production of recombinant proteins since cells do not have to be collected or lysed for the recovery of proteins.

In this study, a novel system for the excretory production of foreign proteins was developed by the use of OmpF fusion system. Outer membrane protein F (OmpF) is a porin protein in the outer membrane of E. coli, and forms the pores through which small hydrophilic molecules diffuse passively. Human □-endorphin composed of 31 amino acids was used as a model protein. The detailed results on the development of a new excretion system and excretory production of human □-endorphin by high cell density culture will be discussed.

본론

Knock-out of ompF gene in E. coli BL21(DE3). For the development of ompF- mutant of E. coli BL21(DE3), red operon of bacteriophage lambda (□) was used and gene replacement was confirmed by PCR. This ompF knock-out mutant was named as E. coli MBEL-BL101 and used as a host cell for the production of OmpF-fused protein.

Construction of ompF gene expression system For the expression of ompF gene, three plasmids, pEDOmpF3, pTrcOmp4, and pOmpF6 were constructed in which the promoters were T7, trc and ompF promoter, respectively. E. coli MBEL-BL101 was transformed with each plasmid and production of OmpF protein into outer membrane were analyized on SDS-PAGE gel (Fig. 1). In E. coli MBEL-BL101 host cell, OmpF protein was not produced and the level of OmpC was increased relatively. In three expression system, pOmpF6 showed the highest level of OmpF production in which ompF gene expression was controlled under ompF promoter. From these results, it was concluded that the expression system using ompF promoter was the most suitable for the efficient production of OmpF-fusion protein.

Fig. 1. Analysis of outer membrane fraction on SDS-PAGE. Lane 1: E. coli BL21(DE3). Lane 2: E. coli MBEL-BL101. Lane 3: E. coli MBEL-BL101 harboring pEDOmpF3. Lane 4: E. coli MBEL-BL101 harboring pTrcOmpF4. Lane 5: E. coli MBEL-BL101 harboring pOmpF6

Extracellular production of OmpF-□-endorphin fusion protein. For the construction of OmpF-□-endorphin fusion gene, pOmpF6E was constructed. In this construct (pOmpF6E), the fused gene can be expressed under the ompF promoter and Factor Xa cleavage site (Ile-Glu-Gly-Arg) was introduced between OmpF and □-endorphin for removal of OmpF protein during the purification. For the excretory production of OmpF-□-endorphin fusion protein, high cell density cultivation was carried out with E. coli MBEL-BL101 harboring pOmpF6E. Fed-batch cultures were carried out in a 6.6 L jar fermentor (Bioflo 3000, New Brunswick Scientific Co., Edison, NJ) containing 2 L of R/2 medium plus 20 g/L glucose. The pH was controlled at 6.8, except for the periods of pH rise due to glucose depletion, by the addition of 28% (v/v) ammonia water. The dissolved oxygen concentration (DOC) was controlled at 40% of air saturation by automatically increasing the agitation speed up to 1,000 rpm and by the change of pure oxygen percentage. Nutrient feeding solution contained 700 g/L of glucose and 20 g/L of MgSO47H2O was added by the pH-stat (with high limit) feeding strategy. During the cultivation, cells grew exponentially and cell dry weight (CDW) reached to 53.3 g/L after 18 hr culture. As cells were grown, the fusion protein was produced into culture supernatant as pure state and the accumulated amount of fusion protein in culture supernatant increased up to 4.53 g/L. From the molar-based calculation, □-endorphin was produced as high as 396 mg/L. Although the purity of OmpF-□-endorphin fusion protein decreased to 45% of total proteins in culture supernatant at the end of fermentation, much higher purity was maintained up to 70% during the exponential growth phase (Fig. 2).

Purification of □-endorphin

The OmpF-□-endorphin fusion protein in the culture supernatant was purified by anion exchange column chromatography. The supernatant was loaded onto an anion-exchange column preequilibrated with 50 mM Tris-HCl (pH 7.0), and then was eluted by a linear gradient of NaCl (0-1.0 M in the same buffer) at 90 mL/h. Protein concentration of each fraction was monitored in a UV detector. After collection of fraction containing the fusion protein, NaCl was removed by dialysis (MWCO 3,500)

Fig 2. SDS-PAGE analysis of proteins in culture supernatant  during high cell density cultivation with E. coli BL101 harboring pOmpF6E.

against 2 L of 20 mM Tris-HCl buffer (pH 7.0) for 24 h with three buffer exchanges. The OmpF protein was cleaved off incubating Factor Xa at 23℃ for 12 h. After cleavage, □-endorphin was finally purified by reverse-phase HPLC using Microsorb-MV C18 column (4.6 x 250 mm) with a linear gradient elution of 100% buffer A to 50% buffer A plus 50% buffer B at 1 mL/min over 2 h [buffer A; 0.1% (v/v) trifluoroacetic acid (TFA); buffer B; acetonitrile containing 0.1 % TFA]. The fraction containing □-endorphin was collected and lyophilized (Fig. 4). The determined sequence of N-terminal amino acid was Tyr-Gly-Gly-Phe-Met-Thr- Ser-Glu-Lys, which was consistent with the N-terminal amino acid sequence of □-endorphin. More than 2.8 mg of pure □-endorphin was obtained from 50 mL of culture supernatant with >99% purity and 13.8% recovery yield. In this study, a new excretion system using OmpF fusion system was development and human □-endorphin was successfully produced into culture medium with high efficiency and purity. Human □-endorphin could be also purified using simple and efficient purification method. The findings that (1) the use of OmpF fusion system for the efficient excretion of recombinant proteins into culture medium and (2) high level production of recombinant protein by high cell density cultivation should be useful for the development of strategies for the efficient extracellular production of other recombinant proteins in E. coli.

Fig. 3. Purification of -endorphin from OmpF fusion protein in culture supernatant. Lanes 1 and 6,  molecular mass standards. Lane 2, the total protein in culture sufernatant. Lane 3, the purified fusion protein using anion exchange column. Lane 4, the □-endorphin cleaved by Factor Xa. Lane 5, the purified □-endorphin using HPLC. The arrow indicates the □-endorphin.

결론

In this study, a new excretion system based on OmpF was developed, and was used to excrete human □-endorphin into the culture medium. Excreted □-endorphin could be purified by simple recovery method. The findings that (1) the OmpF fusion system allowed efficient excretion of recombinant proteins into culture medium, (2) potentially high level production of recombinant protein is possible by high cell density cultivation, and (3) simple recovery is possible because the target protein is excreted into the culture medium, should be useful for the development of strategies for the efficient extracellular production of other recombinant proteins in E. coli.

Acknowledgements

This work was supported by Korean Ministry of Commerce, Industry and Energy (MOCIE) through the Institute of Industrial Technology Policy (ITEP).

참고문헌

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화학공학의 이론과 응용 제8권 제1호 2002년