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FEATURED CASE STUDY
Purification Optimization and Large-Scale Production of a Recombinant Protein from Bacteria for use as a Bio-Pesticide
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Background:

The recombinant protein is a shortened form of the native isolate, requiring multiple tests and regulatory submission characterization. Some bacteria produce toxins that show specific activity against certain insect species. This specificity is mainly due to a unique binding site in the insects' midgut epithelium membrane, where the protein inserts and causes damage to the wall.

Problem/Rational:

This protein has been used extensively as a natural insecticide. However, its purification from bacteria posed a challenge. Previous studies had shown that longer downstream processing times resulted in the truncation of the protein rendering it ineffective for practical use. Hence a cap of 10% truncation was set for clearing a batch for field applications. It was a difficult protein to work with and had issues with storage/stability. The present study focused on developing a robust protocol to meet the specifications.
*Due to the confidentiality clause, we cannot disclose the details of the protein.

Solution/Approach:

The strategy at Premas yielded a rapid protocol and process to purify a recombinant protein pesticide followed by the tag removal using protease with <10% truncation.

Strategy for Purification:

Since time and temperature were the two main factors for protein stability, a detailed plan was strategized for purification where all the steps were overlapped and performed at 4ºC to minimize truncation [Fig.1 and 2].

Purification Procedure:

We applied key modifications in the process parameters to maintain ambient conditions during the protein purification without compromising process touch time and truncation cap of <10%. These parameters include modified protocol for cell lysis, removal of depth filtration, increased centrifugation speed, and modification in IMAC column loading protocol to increase higher residence time for efficient binding. Briefly, cell lysis was done in a high-pressure homogenizer at 900bar with two passes. Continuous ultra-centrifuge at 100,000g with a single pass was used to separate cell debris. The supernatant was then purified using an IMAC column (Figure 3). The protein concentration of eluted fractions was estimated using the Bradford method, and the required protein concentration was maintained. Tag cleaving enzyme was added in the ratio (1:10) based on the amount of protein, and the protein expression was confirmed by immunoblot analysis (Figure 4).

The process developed by Premas successfully decreased the truncation of the protein from 20% to <10% and processing time from 16 to 7.5hrs (Figure 5)


Fig 1: Purification strategy


Fig.2 Procedure for Purification of protein


Fig 3: IMAC purification profile


Fig 4: Protease cleavage of NusA tag A) Before protease addition B) After protease addition


Fig 5: Change in percent truncation with reduced processed time



Measurable Impact:

1) Premas was successful in expressing and purifying recombinant protein pesticide.
2) The process decreased the truncation of protein as a function of reduced processing time, both of which are required for commercial purposes.



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