Protease Treatment-Based Isolation of Chloroplast Inner and Outer Envelope Membranes

The processing, maturation and degradation of proteins in chloroplasts are necessary processes for normal plant growth and development, and are also common regulatory mechanisms in response to environmental changes. Chloroplast proteases play an important role in this process, which can maintain the stability of the protein system in the chloroplast and ensure the orderly progress of the normal life activities of plants. The proteases in the chloroplast are widely distributed, and there are abundant proteases in the envelope, matrix, and thylakoid membranes and chambers, and their functions mainly include: processing and maturation, deletion of abnormal proteins, protein quality control, senescence and plastid type transformation. In conclusion, the various types of proteases contained in the chloroplast constitute a complex and elaborate protease system for maintaining the normal development and function of the chloroplast. As we all know, the premise of making the envelope is to isolate the intact chloroplast. Some studies have found that the inner and outer membranes can be separated by protease treatment.

Fig. 1. Intraplastid proteases in land plants. (Nishimura K, et al., 2016)

Services

In order to understand the properties and functions of each membrane, the inner and outer membranes must be isolated from each other and not affect each other. Protease treatment is one of the common methods for separating chloroplast envelope. For many years, Lifeasible has devoted to developing multiple methods to isolate highly purified chloroplast envelopes, which have been well received by customers around the world. Our scientists have optimized the protocol on the basis of traditional protease treatment, which can achieve rapid and complete separation of chloroplast envelope within 1 day. This approach will greatly facilitate the study of the functional and structural properties of the chloroplast envelope. The process is as follows:

(1) Highly purified chloroplasts were selected for the preparation of membranes.

(2) Intact purified chloroplasts suspended in hypertonic sucrose solution were destroyed by addition of aliquots of trypsin and chymotrypsin.

(3) The envelope is separated from other chloroplast components by flotation centrifugation on a discontinuous density gradient.

(4) The resulting envelope was isolated by isopycnic gradient centrifugation.

(5) Identify the components of the envelope.

Competitive Advantages

• Chloroplast proteases are abundant and readily available.
• Fast separation, can be completed within 1 day.
• The yield and purity of the obtained preparation are high.
• Substrate proteins can only be degraded after entering the central cavity, which avoids non-specific degradation of normal proteins.

Lifeasible can meet the needs of customers on time and on budget through a wide range of chloroplast inner and outer envelope membranes isolation strategies. Our aim is to be customer-centric and to provide the highest quality service to customers around the world. Our skilled and dedicated scientific researchers ensure that the most appropriate methods and techniques are selected for each specialized chloroplast project. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.

Reference

1. Nishimura K, Kato Y, Sakamoto W. (2016) Chloroplast proteases: updates on proteolysis within and across suborganellar compartments[J]. Plant physiology. 171(4): 2280-2293.